Viral vectors containing recombination sites

ABSTRACT

The present invention provides compositions and methods for the construction of nucleic acids comprising all or portion of a viral genome. Nucleic acid molecules of the invention may be constructed to contain multiple recombination and/or topoisomerase recognition sites. The compositions include vectors having multiple recombination sites with unique specificity that contain all or a portion of a viral genome. The methods permit the insertion of a sequence of interest into a viral genome using recombinational and/or topoisomerase-mediated cloning. The present invention also provides methods of constructing recombinant virus, methods of expressing polypeptides, and methods of expressing fusion polypeptides.

BACKGROUND OF THE INVENTION

[0001] 1. Field of the Invention

[0002] The present invention relates to the fields of biotechnology andmolecular biology. In particular, the present invention relates tonucleic acids comprising multiple recombination sites and comprising allor a portion of a viral genome as well as viruses and/or plasmidscontaining multiple recombination sites and their uses.

[0003] 2. Related Art

[0004] Recombinant viruses are currently used in wide variety ofapplications. Viruses may be used for medical applications, for example,in gene therapy applications and/or as vaccines. Viruses may also beused in biotechnology applications, for example, as vectors to clonenucleic acids of interests and/or to produce proteins. Examples ofrecombinant viruses that have been used include, but are not limited to,herpes viruses (see, for example, U.S. Pat. No. 5,672,344, issued toKelly, et al), pox viruses such as vaccinia virus (see, for example,Moss, et al., 1997, in Current Protocols in Molecular Biology, Chapters16.15-16.18, John Wiley & Sons), papilloma viruses (see, for example,U.S. Pat. No. 6,342,224, issued to Bruck, et al.), retroviruses (see,for example U.S. Pat. No. 6,300,118, issued to Chavez, et al.),adenoviruses (see, for example, U.S. Pat. No. 6,261,807, issued toCrouzet, et al.), adeno-associated viruses (AAV, see for example, U.S.Pat. No. 5,252,479, issued to Srivastava), and coxsackie viruses (see,for example, U.S. Pat. No. 6,323,024).

[0005] When the viral nucleic acid is not infectious—for example, poxviruses—construction of recombinant viruses may involve in vivohomologous recombination in a virus-infected cell between the viralgenome and concomitantly transfected plasmid bearing a sequence ofinterest flanked by viral sequences. When the viral nucleic acid isinfectious—for example, adenovirus—a modified viral nucleic acid may beprepared and transfected into a host cell. Either methodology requiresthe preparation of a nucleic acid molecule containing a sequence ofinterest and some or all of the viral sequence. The preparation of thisnucleic acid molecule may be a time-consuming, laborious process.

[0006] Adenoviruses are non-enveloped viruses with a 36 kb DNA genomethat encodes more than 30 proteins. At the ends of the genome areinverted terminal repeats (ITRs) of approximately 100-150 base pairs. Asequence of approximately 300 base pairs located next to the 5′-ITR isrequired for packaging of the genome into the viral capsid. The genomeas packaged in the virion has terminal proteins covalently attached tothe ends of the linear genome.

[0007] The genes encoded by the adenoviral genome are divided into earlyand late genes depending upon the timing of their expression relative tothe replication of the viral DNA. The early genes are expressed fromfour regions of the adenoviral genome termed E1-E4 and are transcribedprior to onset of DNA replication. Multiple genes are transcribed fromeach region. Portions of the adenoviral genome may be deleted withoutaffecting the infectivity of the deleted virus. The genes transcribedfrom regions E1, E2, and E4 are essential for viral replication whilethose from the E3 region may be deleted without affecting replication.The genes from the essential regions can be supplied in trans to allowthe propagation of a defective virus. For example, deletion of the E1region of the adenoviral genome results in a virus that is replicationdefective. Viruses deleted in this region are grown on 293 cells thatexpress the viral E1 genes from the genome of the cell.

[0008] In addition to permitting the construction of a safer,replication-defective viruses, deletion and complementation in trans ofportions of the adenoviral genome and/or deletion of non-essentialregions make space in the adenoviral genome for the insertion ofheterologous DNA sequences. The packaging of viral DNA into a viralparticle is size restricted with an upper limit of approximately 38 kbof DNA. In order to maximize the amount of heterologous DNA that may beinserted and packaged, viruses have been constructed that lack all ofthe viral genome except the ITRs and packaging sequence (see, U.S. Pat.No. 6,228,646). All of the viral functions necessary for replication andpackaging are provided in trans from a defective helper virus that isdeleted in the packaging signal.

[0009] Recombinant adenoviruses have been used as a gene transfervectors both in vitro and in vivo. Their principal attractions as a genetransfer vector are their ability to infect a wide variety of cellsincluding dividing and non-dividing cells and their ability to be grownin cell culture to high titers. A number of systems to insertheterologous DNA into the adenoviral genome have been developed. Theadenoviral genome has been inserted into a yeast artificial chromosome(YAC, see Ketner, et al., PNAS 91:6186-90, 1994). Mutations may beintroduced into the genome by transfecting a mutation-containing plasmidinto a yeast cell that contains the adenoviral YAC. Homologousrecombination between the YAC and the plasmid introduces the mutationinto the adenoviral genome. The adenoviral genome can be removed fromthe YAC by restriction digest and the genome released by restrictiondigest is infectious when transfected into host cells. A similar systemusing two plasmids has been developed in E. coli (see Crouzet, et al.,PNAS 94:1414-1419, 1997, and U.S. Pat. No. 6,261,807). In this system,the adenoviral genome is introduced into a inc-P derived replicon.Mutations are introduced by homologous recombination with a plasmidcontaining a ColE1 origin of replication. The ITRs in the inc-P plasmidare flanked by a restriction site not present in the rest of the viralgenome, thus, infectious DNA can be liberated from the plasmid byrestriction digest.

[0010] A number of viruses containing recombination site sequencesand/or encoding recombinases have been prepared. For example, the Crerecombinase has been expressed from recombinant adenovirus and used toexcise fragments from a mouse genome that were flanked with lox sites(see, Wang, et al., PNAS 93:3932-3936, 1996). U.S. Pat. No. 6,156,497describes a system for constructing adenoviral genomes utilizing a firstnucleic acid having an ITR, packaging signal, DNA of interest, andrecombination site and a second nucleic acid having a recombination siteand an ITR to which is bound a terminal protein. In the presence ofrecombinase, the two molecules are joined to form an infectious viralDNA.

[0011] Baculoviruses are large, enveloped viruses that infectarthropods. Baculoviral genomes are double-stranded DNA molecules ofapproximately 130 kbp in length. Baculoviruses have gained widespreaduse as systems in which to express proteins, particularly proteins fromeukaryotic organisms (e.g., mammals), as the insect cells used toculture the virus may more closely mimic the post-translationalmodifications (e.g., glycosylation, acylation, etc.) of the nativeorganism.

[0012] Numerous expression systems utilizing recombinant baculoviruseshave been developed. General methods for constructing recombinantbaculoviruses for expression of heterologous proteins may be found inPiwnica-Worms, et al., (1997) Expression of Proteins in Insect CellsUsing Baculovirus Vectors, in Current Protocols in Molecular Biology,Chapter 16, pp. 16.9.1 to 16.11.12, Ausubel, et al. Eds., John Wiley &Sons, Inc. Other expression systems are known, for example, U.S. Pat.No. 6,255,060, issued to Clark, et al. discloses a baculoviralexpression system for expressing nucleotide sequences that include atag. U.S. Pat. No. 5,244,805, issued to Miller, discloses a baculoviralexpression system that utilizes a modified promoter not naturally foundin baculoviruses. U.S. Pat. No. 5,169,784, issued to Summers, et al.discloses a baculoviral expression system that utilizes dual promoters(e.g., a baculoviral early promoter and a baculoviral late promoter).U.S. Pat. No. 5,162,222, issued to Guarino, et al. discloses abaculoviral expression system that can be used to create stable cellslines or infectious viruses expressing heterologous proteins from abaculoviral immediate-early promoter (i.e., IEN). U.S. Pat. No.5,155,037, issued to Summers, et al. discloses a baculoviral expressionsystem that utilizes insect cell secretion signal to improve efficiencyof processing and secretion of heterologous genes. U.S. Pat. No.5,077,214, issued to Guarino, et al. discloses the use of baculoviralearly gene promoters to construct stable cell lines expressionheterologous genes. U.S. Pat. No. 4,879,239, issued to Smith, et al.discloses a baculoviral expression system that utilizes the baculoviralpolyhedrin promoter to control the expression of heterologous genes.

[0013] Various methods of constructing recombinant baculoviruses havebeen used. A frequently used method involves transfecting baculoviralDNA and a plasmid containing baculoviral sequences flanking aheterologous sequence. Homologous recombination between the plasmid andthe baculoviral genome results in a recombinant baculovirus containingthe heterologous sequences. This results in a mixed population ofrecombinant and non-recombinant viruses. Recombinant baculoviruses maybe isolated from non-recombinant by plaque purification. Virusesproduced in this fashion may require several rounds of plaquepurification to obtain a pure strain. Methods to reduce the backgroundof non-recombinant viruses produced by homologous recombination methodshave been developed. For example, a linearized baculoviral genomecontaining a lethal deletion, BaculoGold™, is commercially availablefrom BD Biosciences, San Jose, Calif. The lethal deletion is rescued byhomologous recombination with plasmids containing baculoviral sequencesfrom the polyhedrin locus.

[0014] Methods utilizing direct insertion of foreign sequences into abaculoviral genome are also known. For example, Peakman, et al. (NucleicAcids Res 20(3):495-500, 1992) disclose the construction ofbaculoviruses having a lox site in the genome. Heterologous sequencesmay be moved into the genome by in vitro site-specific recombinationbetween a plasmid having a lox site and the baculoviral genome in thepresence of Cre recombinase. U.S. Pat. No. 5,348,886, issued to Lee, etal. discloses a baculoviral expression system that utilizes a bacmid (ahybrid molecule comprising a baculoviral genome and a prokaryotic originof replication and selectable marker) containing a recombination sitefor Tn7 transposon. Prokaryotic cells carrying the bacmid aretransformed with a plasmid having a Tn7 recombination site and with aplasmid expressing the activities necessary to catalyze recombinationbetween the Tn7 sites. Heterologous sequences present on the plasmid areintroduced into the bacmid by site-specific recombination between theTn7 sites. The recombinant bacmid may be purified from the prokaryotichost and introduced into insect cells to initiate an infection.Recombinant viruses carrying the heterologous sequence are produced bythe cells transfected with the bacmid.

[0015] The family Retroviridae contains three subfamilies: 1)oncovirinae; 2) spumavirinae; and 3) lentivirinae. Retroviruses (e.g.,lentiviruses) are viruses having an RNA genome that replicate through aDNA intermediate. A retroviral particle contains two copies of the RNAgenome and viral replication enzymes in a RNA-protein viral core. Thecore is surrounded by a viral envelop made up of virally encodedglycoproteins and host cell membrane. In the early steps of infection,retroviruses deliver the RNA-protein complex into the cytoplasm of thetarget cell. The RNA is reverse transcribed into double-stranded cDNAand a pre-integration complex containing the cDNA and the viral factorsnecessary to integrate the cDNA into the target cell genome is formed.The complex migrates to the nucleus of the target cell and the cDNA isintegrated into the genome of the target cell. As a consequence of thisintegration, the DNA corresponding to the viral genome (and anyheterologous sequences contained in the viral genome) is replicated andpassed on to daughter cells. This makes it possible to permanentlyintroduce heterologous sequences into cells.

[0016] A wide variety of retroviruses are known, for example, leukemiaviruses such as a Moloney Murine Leukemia Virus (MMLV) andimmunodeficiency viruses such as the Human Immunodeficiency Virus (HIV).Representative examples of retroviruses include, but are not limited to,the Gibbon Ape Leukemia virus (GALV), Avian Sarcoma-Leukosis Virus(ASLV), which includes but is not limited to Rous Sarcoma Virus (RSV),Avian Myeloblastosis Virus (AMV), Avian Erythroblastosis Virus (AEV)Helper Virus, Avian Myelocytomatosis Virus, Avian ReticuloendotheliosisVirus, Avian Sarcoma Virus, Rous Associated Virus (RAV), andMyeloblastosis Associated Virus (MAV).

[0017] Retroviruses have found widespread use as gene therapy vectors.To reduce the risk of transmission of the gene therapy vector, genetherapy vectors have been developed that have modifications that preventthe production of replication competent viruses once introduced into atarget cell. For example, U.S. Pat. No. 5,741,486 issued to Pathak, etal. describes retroviral vectors comprising direct repeats flanking asequence that is desired to be deleted (e.g., a cis-acting packingsignal) upon reverse transcription in a host cell. Deletion of thepacking signal prevents packaging of the recombinant viral genome intoretroviral particles, thus preventing spread of retroviral vectors tonon-target cells in the event of infection with replication competentviruses. U.S. Pat. Nos. 5,686,279, 5,834,256, 5,858,740, 5,994,136,6,013,516, 6,051,427, 6,165,782, and 6,218,187 describe a retroviralpackaging system for preparing high titer stocks of recombinantretroviruses. Plasmids encoding the retroviral functions required topackage a recombinant retroviral genome are provided in trans. Thepackaged recombinant retroviral genomes may be harvested and used toinfect a desired target cell.

[0018] The family Herpesviridae contains three subfamilies 1)alphaherpesvirinae, containing among others human herpesvirus 1; 2)betaherpesvirinae, containing the cytomegaloviruses; and 3)gammaherpesvirinae. Herpesviruses are enveloped DNA viruses.Herpesviruses form particles that are approximately spherical in shapeand that contain one molecule of linear dsDNA and approximately 20structural proteins. Numerous herpesviruses have been isolated from awide variety of hosts. For example, U.S. Pat. No. 6,121,043 issued toCochran, et al. describes recombinant herpesvirus of turkeys comprisinga foreign DNA inserted into a non-essential region of the herpesvirus ofturkeys genome; U.S. Pat. No. 6,410,311 issued to Cochran, et al.describes recombinant feline herpesvirus comprising a foreign DNAinserted into a region corresponding to a 3.0 kb EcoRI-SalI fragment ofa feline herpesvirus genome, U.S. Pat. No. 6,379,967 issued to Meredith,et al., describes herpesvirus saimiri, (HVS; a lymphotropic virus ofsquirrel monkeys) as a viral vector; and U.S. Pat. No. 6,086,902 issuedto Zamb, et al. describes recombinant bovine herpesvirus type 1vaccines.

[0019] Herpesviruses have been used as vectors to deliver exogenousnucleic acid material to a host cell. In addition to the examples above,U.S. Pat. No. 4,859,587, issued to Roizman describes recombinant herpessimplex viruses, vaccines and methods, U.S. Pat. No. 5,998,208 issued toFraefel, et al., describes a helper virus-free herpesvirus vectorpackaging system, U.S. Pat. No. 6,342,229 issued to O'Hare, et al.,describes herpesvirus particles comprising fusion protein and theirpreparation and use and U.S. Pat. No. 6,319,703 issued to Speckdescribes recombinant virus vectors that include a double mutantherpesvirus such as an herpes simplex virus-1 (HSV-1) mutant lacking theessential glycoprotein gH gene and having a mutation impairing thefunction of the gene product VP 16.

[0020] RNA viruses, such as those of the families Flaviviridae andTogaviridae have also been used to deliver exogenous nucleic acids totarget cells. For example, members of the genus alphavirus in the familyTogaviridae have been engineered for the high level expression ofheterologous RNAs and polypeptides (Frolov et al., Proc. Natl. Acad.Sci. U.S.A. 93: 11371-11377 (1996)). Alphaviruses are positive strandedRNA viruses. A single genomic RNA molecule is packaged in the virion.RNA replication occurs by synthesis of a full-length minus strand RNAintermediate that is used as a template for synthesis of positive strandgenomic RNA as well for synthesis of a positive strand sub-genomic RNAinitiated from an internal promoter. The sub-genomic RNA can accumulateto very high levels in infected cells making alphaviruses attractive astransient expression systems. Examples of alphaviruses are Sindbis virusand Semliki Forest Virus. Kunjin virus is an example of a flavivirus.Sub-genomic replicons of Kunjin virus have been engineered to expressheterologous polypeptides (Khromykh and Westaway, J. Virol. 71:1497-1505 (1997)). The genomic RNA of both flaviviruses and togavirusesare infectious; transfection of the naked genomic RNA results inproduction of infective virus particles.

[0021] Methods for constructing recombinant viruses are typicallylaborious and time consuming. There remains a need in the art formaterials and methods for the rapid and precise and rapid constructionof recombinant viruses containing a nucleic acid region of interest.This need and others are met by the present invention.

BRIEF SUMMARY OF THE INVENTION

[0022] The present invention provides, in part, a nucleic acid moleculecomprising all or a portion of a viral genome (e.g., an adenovirusgenome, a baculovirus genome, a herpesvirus genome, a pox virus genome,an adeno-associated virus genome, a retrovirus genome, a flavivirusgenome, a togavirus genome, an alphavirus genome, an RNA virus genome,etc.). Nucleic acid molecules of the invention may further comprise atleast two recombination sites (e.g., three, four, five, six, seven,eight, nine, ten, etc.) that, in most instances, do not recombine witheach other. In particular embodiments, the viral genome may be anadenoviral genome, a baculoviral genome, a retroviral genome (e.g., alentiviral genome), an RNA virus genome or a herpesvirus genome. In someembodiments, the viral genome is not an adenoviral genome, is not abaculoviral genome, is not a retroviral genome (e.g., a lentiviralgenome), and/or is not a herpesvirus genome. In some embodiments, theviral genome is not from a virus that infects prokaryotic organisms. Insome embodiments, one or more of the two or more recombination sites isnot a lox site. In some embodiments, nucleic acid molecules comprisingone or more sequences of interest are combined with nucleic acidmolecules comprising all or a portion of a viral genome using arecombination system that does not use a recombination system derivedfrom a transposon (e.g., Tn7). In some embodiments, nucleic acidmolecules of the invention may not contain a lox site.

[0023] Optionally, nucleic acid molecules of the invention may compriseone or more features that confer desired characteristics on the nucleicacid molecules. Examples of features include, but are not limited to,promoters, viral terminal repeats (e.g., long terminal repeats (LTRs)),splice sites (e.g., 5′-splice doneor sites and/or 3′-splice acceptorsites), packaging signals, nucleic acid sequences responsive to one ormore viral proteins (e.g., rev response element (RRE)), recognitionsites (e.g., restriction enzyme recognition sites), recombination sites,sequences encoding marker proteins or polypeptides (e.g., antibioticresitance enzymes, toxic proteins, etc.), sequences encoding epitopesrecognizable by an antibody (e.g., V5 epitope), origins of replication(which may function in prokaryotic and/or eukaryotic cells), interveningsequences (e.g., β-globin intron), internal ribosome entry sequences(IRES), and polyadenylation signals (e.g., SV40 polyadenylation signal).Additional examples of such nucleic acid molecules include those whichcontain at least (1) one or more (e.g., one, two, three, four, five,six, seven, eight, nine, etc.) component of one or more of the vectorsrepresented in FIGS. 1, 2, 4, 5, 6, 7, 8, 9, 10, 15, 18, 20, 22, 34, 36,37, 49, 57, 58, 59, 60, 69, 70, 71 or 72; or (2) one or more componentsof such vectors which confer the same or similar feature upon a nucleicacid molecule. As a specific example, a nucleic acid molecule of theinvention may be a vector which comprises, in addition to recombinationsites, at least one blasticidin resistance marker (see, e.g., FIG. 22),at least one GP64 promoter (see, e.g., FIG. 22), at least one RSVpromoter (see, e.g., FIG. 36A), at least one beta-globin intron (see,e.g., FIG. 37A), at least one ampicillin resistance marker (see, e.g.,FIG. 37A), and at least one bacterial origin of replication (see, e.g.,FIG. 37A). In most instances, the combinations of components selectedfor inclusion in a nucleic acid molecule will be designed to provideactivities intended for a particular use. For example, a vector which iscapable of expressing a nucleic acid insert in more than one type ofeukaryotic cells (e.g., human cells and insect cells) and is replicablein prokaryotic cells (e.g., E. coli cells) may be desired. Thus, thecomponents which are selected for inclusion in nucleic acid molecules ofthe invention will typically be determined by the particular use forwhich it is designed. The invention further includes methods for makingand using such nucleic acid molecules as described, for example,elsewhere herein.

[0024] Viruses produced using nucleic acids of the present invention maybe used as viral vectors (e.g., viruses containing at least oneheterologous sequence), for example, to deliver exogenous sequences tocells or organisms. The present invention also contemplates compositionscomprising nucleic acids and/or viruses of the invention, as well asmethods of making and using such nucleic acids, viruses, andcompositions.

[0025] Viral genomes that may be used with the present invention (e.g.,retroviral genomes, adenoviral genomes, herpesvirus genomes, genomes ofRNA viruses, and/or baculoviral genomes) may be wild type or may containone or more mutations, insertions and/or deletions. In some embodiments,viral genomes for use in the practice of the present invention may beadenoviral genomes containing one or more deletions. Deleted adenoviralgenomes may be deleted in one or more regions of the genome. Regions ofthe adenoviral genome that may be deleted, include, but are not limitedto, the E1 and E3 regions.

[0026] Adenoviral genomes for use in the present invention may beinfectious. In some embodiments, an adenoviral genome may be infectiouswhen introduced into cells expressing one or more adenoviral proteins(e.g., the E1 proteins as in 293 cells). In some embodiments, a viralgenome used in the invention is an Ad5 viral genome.

[0027] Baculoviral genomes that may be used in the practice of thepresent invention may be entire genomes or may contain one or moredeletions, for example, at the polyhedrin locus. Suitable genomesinclude those from any virus in the family Baculoviridae. Suitable viralgenomes include, but are not limited to, those from occludedbaculoviruses (e.g., nuclear polyhedrosis viruses (NPV) such asAutographa californica nuclear polyhedrosis virus (AcMNPV),Choristoneura fumiferana MNPV (CfMNPV), Mamestra brassicae MNPV(MbMNPV), Orgyia pseudotsugata MNPV (OPMNPV), Bombyx mori S NuclearPolyhedrosis Virus (BmNPV), Heliothis zea SNPV (HzSnpv), andTrichoplusia ni SNPV (TnSnpv) and granulosis viruses (GV) (e.g., Plodiainterpunctella granulosis virus (PiGV), Trichoplusia ni granulosis virus(TnGV), Pieris brassicae granulosis virus (PbGV), Artogeia rapaegranulosis virus (ArGV), and Cydia pomonella granulosis virus (CpGV)).Suitable genomes also include, but are not limited to, those fromnon-occluded baculoviruses (NOB) (e.g., Heliothis zea NOB (HzNOB),Oryctes rhinoceros virus), etc.

[0028] In some embodiments, viral genomes for use in the practice of thepresent invention may be retroviral genomes containing one or moredeletions. Deleted retroviral genomes may be deleted in one or moreregions of the genome. Regions of the retroviral genome that may bedeleted, include, but are not limited to, the gag, pol, env, and revregions. In some embodiments, a retroviral genome may be deleted of allretroviral sequences except the 5′-LTR, 3′-LTR and packaging signal (Ψ).In some embodiments, retroviral genomes of the present invention maycomprise one or more heterologous sequences (e.g., sequences derivedfrom another organism such as another virus). In a particularembodiment, nucleic acid molecules of the invention may comprise adeleted retroviral genome and may also comprise one or more heterologoussequences that may be promoter sequences. In some embodiments, nucleicacid molecules of the invention may comprise a deleted retroviral genomeand may further comprise the CMV promoter.

[0029] In some embodiments, nucleic acid molecules of the presentinvention may be in the form of plasmids and/or bacmids comprising oneor more origins of replication and, optionally, one or more selectablemarkers. In certain embodiments, nucleic acid molecules of the invention(e.g., plasmids and/or bacmids) may comprise one or more recognitionsequences (e.g., recombination sequences, topoisomerase sequences,restriction enzyme sequences, etc.), which may be recognized by the sameor different enzymes. For example, in some embodiments, plasmidscomprising all or a portion of the viral genome may comprise one or morerecombination sites that may not recombine with each other. In certainembodiments, nucleic acid molecules of the invention (e.g., plasmidsand/or bacmids) may comprise restriction enzyme recognition sequences,which may be recognized by the same or different restrictionendonucleases, arranged such that digestion with one or more restrictionenzymes that recognize the recognition sequences produces a linearmolecule comprising the viral genome. In some embodiments, digestionwith a restriction enzyme may remove a portion of plasmid and/or bacmid.For example, in some embodiments, plasmids comprising all or a portionof the adenoviral genome may be digested so as to remove the origin ofreplication and, optionally, the selectable marker from the plasmid. Inanother example, a nucleic acid molecule comprising all or a portion ofa baculoviral genome may be digested with a restriction enzyme thatlinearizes the baculoviral genome, for example, by cleaving the nucleicacid molecule at a recognition site located between two recombinationsites (see FIG. 20). In embodiments of this type, the baculoviral genomemay be re-circularized by recombination with a second nucleic acidmolecule having recombination sites that are capable of recombining withthose in the nucleic acid molecule comprising all or a portion of thebaculoviral genome. In particular embodiments, the restriction enzymerecognition sites may be recognized by two different restrictionenzymes. Thus, the invention includes methods for selecting recombinantnucleic acid molecules (e.g., recombinant baculoviral vectors). Themethod may comprise recombining a first nucleic acid molecule, which maybe linearized, with a second nucleic acid molecule to produce acircularized molecule that is capable of replicating when introducedinto a suitable host cell. The method may also comprise selectingagainst re-circularized first nucleic acid molecule that did not undergorecombination with the second nucleic acid molecule. In someembodiments, the first nucleic acid molecule may be a linearizedbaculoviral genome.

[0030] A nucleic acid sequence of interest may be inserted into thenucleic acid molecule of the invention using recombinational cloningtechniques. In some embodiments, a nucleic acid molecule of theinvention may comprise a heterologous promoter (e.g., the CMV promoter)and one or more recombination sites arranged such that a nucleic acidsequence of interest can be inserted into the nucleic acid molecule ofthe invention by recombination with one or more of the recombinationsites and, after insertion, the nucleic acid sequence of interest may beoperably linked to the heterologous promoter. In some embodiments, anucleic acid molecule of the invention may have a heterologous promoterlocated adjacent to two recombination sites that do not recombine witheach other. A nucleic acid sequence of interest can be inserted into thenucleic acid molecule of the invention between the two recombinationsites and may then be operably linked to the heterologous promoter.

[0031] Any nucleic acid sequence of interest may be placed between therecombination sites present in the nucleic acids of the presentinvention. For example, the nucleic acid sequence between therecombination sites may encode one or more polypeptides of interest. Theviral vectors of the present invention may be used to express librariesof sequences, for example, genomic libraries or cDNA libraries. Asequence of interest may be a sequence coding for a polypeptide or maybe a sequence that does not encode a polypeptide. Examples of sequencesof interest that do not encode a polypeptide include, but are notlimited to, sequences encoding tRNA sequences (e.g., suppressor tRNAsequences), sequences encoding ribozyme sequences, promoter sequences,enhancer sequences, repressor sequences and the like. In someembodiments, the sequence of interest may encode one or morepolypeptides and may further comprise one or more stop codons in thesequence. In some embodiments, the nucleic acid between therecombination sites comprises at least one selectable marker. In someembodiments, the sequence of interest comprises a sequence encoding atleast one suppressor tRNA and/or at least one aminoacyl-tRNA synthetase.

[0032] In some embodiments, the present invention provides nucleic acidmolecules comprising all or a potion of more than one viral genome. Forexample, a nucleic acid molecule of the invention may comprise all or aportion of a first viral genome (e.g., a retroviral genome) and all or aportion of one or more additional viral genomes (e.g., an adenoviralgenome, a baculoviral genome, a herpesvirus genome, a pox virus genome,an RNA virus genome, etc). In some embodiments, the nucleic acidmolecules of the invention may comprise nucleic acid sequences from morethan one virus. Nucleic acid molecules of this type may comprise viralsequences that permit the replication of the nucleic acid in more thanone type of organism (e.g., mammalian cells and insect cells) and mayalso include sequences capable of functioning as transcriptionalregulatory sequences (e.g., promoters, enhancers, etc.) that function inmore than one cell type. For example, one viral sequence may function asa promoter in one cell type (e.g., mammalian) while another viralsequence may function as a promoter in another cell type (e.g., insect).

[0033] In another aspect, the present invention provides a method ofconstructing a nucleic acid molecule comprising all or a portion of oneor more viral genomes (e.g., a recombinant virus such as a viralvector). In some embodiments, methods of the invention may compriseproviding at least a first nucleic acid molecule comprising all or aportion of at least one viral genome and at least a first and a secondrecombination site that do not recombine with each other. Methods of theinvention may also entail contacting at least a first nucleic acidmolecule with at least a second nucleic acid molecule comprising atleast one sequence of interest flanked by at least a third and a fourthrecombination site under conditions causing recombination between thefirst and third recombination site and between the second and fourthrecombination site. In some embodiments, the viral genome may be anadenoviral genome, for example, an Ad5 adenoviral genome. In someembodiments, the viral genome may be a baculoviral genome, for example,an Autographa califomica multiple nuclear polyhedrosis virus (ACMNPV)genome. In some embodiments, the viral genome may be a retroviral genome(e.g., a lentiviral genome).

[0034] In some embodiments, a first nucleic acid molecule comprising allor a portion of a viral genome for use in the methods of the inventionmay be a plasmid that may comprise an origin of replication and aselectable marker. The first nucleic acid molecule may, optionally,contain two restriction enzyme recognition sequences, which may be forthe same or different restriction enzymes, arranged such that digestionwith the appropriate restriction enzyme or restriction enzymes producesa linear molecule comprising the viral genome (e.g., adenoviral genome)and lacking the origin of replication and/or the selectable marker.

[0035] In some embodiments, the first nucleic acid molecule may compriseat least a first and a second recombination site, which may or may notrecombine with each other, and the portion of the first nucleic acidmolecule between the first and second recombination sites may comprise asequence encoding at least one selectable marker. In some embodiments, asecond nucleic acid molecule, which may or may not comprise viralsequences, may comprise at least a third and a fourth recombination siteand a sequence of interest between the third and fourth recombinationsite. The sequence of interest may be any sequence, for example, asequence encoding a polypeptide or a sequence of a functional RNA (e.g.,a suppressor tRNA sequence). In some embodiments, the first and secondnucleic acid molecules may be contacted with one or more recombinationproteins such that the sequence of interest is transferred to the firstnucleic acid molecule resulting in a first nucleic acid moleculecomprising all or a portion of a viral genome and further comprising atleast one sequence of interest (e.g., a polypeptide coding region, atRNA coding sequence etc.). The present invention also contemplatescompositions comprising a nucleic acid molecule comprising all or aportion of a viral genome and further comprising at least one sequenceof interest, as well as methods of making and using such nucleic acidsand compositions. In some embodiments, the sequence of interest may be atRNA coding sequence.

[0036] In some embodiments, a first nucleic acid molecule comprising allor a portion of a viral genome for use in the methods of the inventionmay be a bacmid that may comprise an origin of replication and aselectable marker. The first nucleic acid molecule may, optionally,contain a restriction enzyme recognition sequence, located such thatdigestion with the appropriate restriction enzyme produces a linearmolecule comprising the viral genome (e.g., baculoviral genome). In someembodiments, the first nucleic acid molecule may comprise at least afirst and a second recombination site, which may or may not recombinewith each other, and the recognition site for the restriction enzyme maybe located between the recombination sites. Optionally, the portion ofthe first nucleic acid molecule between the first and secondrecombination sites may comprise a sequence encoding at least oneselectable marker. In some embodiments, a second nucleic acid molecule,which may or may not comprise viral sequences, may comprise at least athird and a fourth recombination site and the sequence between the thirdand fourth recombination site comprises a sequence of a functional RNA(e.g., a suppressor tRNA sequence). In some embodiments, the first andsecond nucleic acid molecules may be contacted with one or morerecombination proteins such that the functional sequence (e.g., asequence encoding a suppressor tRNA sequence) is transferred to thefirst nucleic acid molecule resulting in the first nucleic acid moleculere-circularizing and further comprising at least one functional sequence(e.g., a sequence encoding a tRNA). The present invention alsocontemplates compositions comprising a nucleic acid molecule comprisingall or a portion of a viral genome and further comprising at least onefunctional sequence, as well as methods of making and using such nucleicacids and compositions.

[0037] The present invention also provides, in part, materials andmethods for joining or combining two or more (e.g., two, three, four,five, seven, ten, twelve, fifteen, twenty, thirty, fifty, seventy-five,one hundred, two hundred, etc.) nucleic acid segments and/or nucleicacid molecules by a recombination reaction between recombinationsites—at least one of which is present on each molecule and/orsegment—in order to construct a nucleic acid molecule comprising all ora portion of a viral genome (e.g., a retroviral genome, an adenoviralgenome and/or a baculoviral genome). In embodiments of this type, one ormore nucleic acid segments and/or nucleic acid molecules may compriseviral nucleic acid sequences. Such recombination reactions to joinmultiple nucleic acid segments and/or nucleic acid molecules accordingto the invention may be conducted in vivo (e.g., within a cell, tissue,organ or organism) or in vitro (e.g., cell-free systems). The inventionalso relates to hosts and host cells comprising the viral vectors and/ornucleic acid molecules of the invention. The invention also relates tokits for carrying out methods of the invention, and to compositions forcarrying out methods of the invention, as well as to compositions usedin and made while carrying out the methods of the invention.

[0038] Nucleic acid molecules prepared by methods of the invention maybe used for any purpose known to those skilled in the art. For example,nucleic acid molecules of the invention may be used to express proteinsor peptides encoded by these nucleic acid molecules and may also be usedto create novel fusion proteins by expressing different nucleic acidsequences linked by the methods of the invention. Nucleic acids of theinvention may also be used to produce RNA molecules that are nottranslated into polypeptides or proteins, for example, tRNAs, anti-sensemolecules, interfering RNA and/or ribozymes.

[0039] Nucleic acid molecules of the invention may be used as part of asystem to generate replication-defective viral particles. For example,nucleic acid molecules of the invention may be packaged into a viralparticle using techniques known in the art. Packaging may beaccomplished by providing requisite packaging activities in trans, forexample, on a different nucleic acid molecule and/or in the genome of acell. In a particular example, nucleic acid molecules of the inventionmay be used to construct a replication-defective lentivirus. In aparticular embodiment, nucleic acid molecules of the invention maycomprise lentiviral long terminal repeats and packaging signal and otheractivities required to package the nucleic acid molecule of theinvention may be provided in trans, for example, may be expressed fromone or more plasmids.

[0040] In some aspects, methods of the present invention may compriseintroducing a nucleic acid molecule of the invention into a cell orpopulation of cells and detecting the presence or absence of the nucleicacid molecule. Such detection may be accomplished, for example, bydetecting the presence or absence of one or more selectable markerpresent on the nucleic acid molecule. Optionally, a selectable markermay be a nucleic acid sequence encoding a polypeptide having β-lactamaseactivity. Detection may be accomplished by contacting a cell orpopulation of cells with a fluorogenic substrate for β-lactamaseactivity and detecting fluorescence of the cell or population of cells.In a specific embodiment, the fluorogenic substrate may be CCF2/AM andfluorescence may be detected by illuminating the cell with light havinga wavelength of 405 nm and detecting fluorescence at a wavelength ofapproximately 450 nm and at a wavelength of approximately 520 nm.Methods may also comprise comparing the amount of fluorescence observedat 450 nm and 520 mn, for example, by determining a ratio between theobserved fluorescence amounts. Methods may also comprise physicallyseparating cells having a desired nucleic acid molecule by fluorescentactivated cell sorting (FACS).

[0041] The present invention provides methods for infecting,transfecting, transducing and/or otherwise introducing the nucleic acidmolecules of the invention into host cells and, optionally, expressingone or more sequences of interest present on the nucleic acid moleculeof the invention. Suitable host cells may be dividing or non-dividingcells. In a particular embodiment, host cells using in connection withthe methods of the invention are non-dividing cells. For example, one ormore nucleic acid molecule of the invention may be introduced into oneor more non-dividing cells. One or more of the nucleic acid moleculesmay comprise a sequence of interest that may encode a polypeptide or anuntranslated RNA. The methods of the invention may result in theproduction in the non-dividing cells of a polypeptide or untranslatedRNA encoded by the sequence of interest. Nucleic acid molecules of theinvention for use in the expression of a sequence of interest in anon-dividing cell may comprise one or more sequences from one or moreviruses, for example, from an adenovirus and/or a lentivirus. A nucleicacid molecule of the invention for expression of a sequence of interestin a non-dividing cell may comprise one or more adenoviral sequences. Anucleic acid molecule of the invention for expression of a sequence ofinterest in a non-dividing cell may comprise one or more lentiviralsequences.

[0042] Recombination sites for use in the methods and/or compositions ofthe invention may be any recognition sequence on a nucleic acid moleculethat participates in a recombination reaction mediated or catalyzed byone or more recombination proteins. In those embodiments of the presentinvention utilizing more than one (e.g., two, three, four, five, seven,ten, twelve, fifteen, twenty, thirty, fifty, etc.) recombination sites,such recombination sites may be the same or different and may recombinewith each other or may not recombine or not substantially recombine witheach other. Recombination sites contemplated by the invention alsoinclude mutants, derivatives or variants of wild-type or naturallyoccurring recombination sites. Desired modifications can also be made tothe recombination sites to include changes to the nucleotide sequence ofthe recombination site that cause desired sequence changes to thetranscription product (e.g., mRNA, tRNA, ribozyme, etc.) and/or desiredamino acid changes in the translation product (e.g., polypeptide orprotein) when transcription occurs across the modified recombinationsite.

[0043] Preferred recombination sites used in accordance with theinvention include att sites, frt sites, dif sites, psi sites, cer sites,and lox sites or mutants, derivatives and variants thereof (orcombinations thereof). Recombination sites contemplated by the inventionalso include portions of such recombination sites. Depending on therecombination site specificity used, the invention allows directionallinking of nucleic acid molecules to provide desired orientations of thelinked molecules or non-directional linking to produce randomorientations of the linked molecules.

[0044] In certain embodiments, recombination proteins used in thepractice of the invention comprise one or more proteins selected fromthe group consisting of Cre, Int, IHF, Xis, Flp, Fis, Hin, Gin, Cin, Tn3resolvase, TndX, XerC, XerD, and ΦC31. In specific embodiments, therecombination sites comprise one or more recombination sites selectedfrom the group consisting of lox sites; psi sites; dif sites; cer sites;fit sites; att sites; and mutants, variants, and derivatives of theserecombination sites that retain the ability to undergo recombination.

[0045] In a specific aspect, the invention provides nucleic acidmolecules and/or viral vectors that permit controlled expression offusion polypeptides by suppression of one or more stop codons. Accordingto the invention, a nucleic acid molecule, which may be any nucleic acidmolecule, for example, a plasmid and/or a nucleic acid moleculecomprising all or a portion of a viral genome and/or a viral vectorproduced by the methods of the invention, may comprise a sequence ofinterest that may comprise one or more stop codons (e.g., TAG, TAA,and/or TGA) that may be suppressed. In embodiments of this type, mRNA istranscribed from the nucleic acid molecule. The transcribed mRNAmolecule comprises at least a first coding sequence corresponding to thesequence of interest and at least one additional sequence containing asecond coding region separated from the first coding sequence by a stopcodon. Suppression of the stop codon allows expression of both the firstand second coding sequences in a single polypeptide molecule. Thenucleic acid sequence corresponding to the additional sequence may becontained on the sequence of interest or may be contained in arecombination site or on the nucleic acid molecule. One or moresuppressor tRNA molecules may be provided, for example, from any nucleicacid molecule such as a plasmid, a nucleic acid molecule comprising allor a portion of a viral genome and/or a viral vector of the invention.

[0046] Some embodiments of the present invention allow selective orcontrolled fusion protein expression by varying the suppression ofselected stop codons. For example, a nucleic acid molecule, which may bea viral vector of the invention, may comprise three coding regions ofinterest separated by regions comprising stop codons. One or more of thecoding regions of interest may be flanked by recombination sites. Bysuppressing the stop codon between the first and second coding regions afusion polypeptide may be produced comprising amino acids encoded by thefirst and second coding region but not containing the amino acidsencoded by the third region. Thus, use of different stop codons andvariable control of suppression allows production of various fusionproteins or portions thereof encoded by all or different portions of thenucleic acid sequence of interest. In some embodiments, one or more ofthe coding regions in the sequence of interest may encode a polypeptidethat comprises a sequence (preferably an N-terminal and/or a C-terminaltag sequence) encoding all or a portion of one or more of the following:the Fc portion of an immunoglobin, an antibody, a β-glucuronidase, aβ-lactamase, a β-galactosidase, a fluorescent protein (e.g., greenfluorescent protein, yellow fluorescent protein, red fluorescentprotein, cyan fluorescent protein, etc.), a transcription activationdomain, a protein or domain involved in translation, proteinlocalization tag, a protein stabilization or destabilization sequence, aprotein interaction domains, a binding domain for DNA, a proteinsubstrate, a purification tag (e.g., an epitope tag, maltose bindingprotein, a six histidine tag, glutathione S-transferase, etc.), and anepitope tag.

[0047] In one aspect, a stop codon may be included anywhere within thesequence of interest or within a recombination site contained by nucleicacid molecules, which may be nucleic acid molecules comprising all or aportion of a viral genome. Preferably, stop codons are located at ornear the termini of the sequence of interest, although stop codons maybe included internally within the sequence. In another aspect, thesequence of interest may comprise the coding sequence of all or aportion of a target gene or open reading frame (ORF) of interest whereinthe coding sequence is followed by a stop codon. The stop codon may thenbe followed by a recombination site allowing joining the sequence ofinterest to another nucleic acid molecule, which may be a nucleic acidmolecule comprising all or a portion of a viral genome. After joiningthe sequence of interest with the nucleic acid molecule to form arecombinant nucleic acid molecule, the stop codon may be optionallysuppressed by a suppressor tRNA molecule. In some embodiments of thistype, one or more genes coding for one or more suppressor tRNA molecules(that may be the same or different) may be provided on the same nucleicacid molecule, or on another nucleic acid molecule. One or more genescoding for one or more suppressor tRNA molecules (that may be the sameor different) may be provided on a different nucleic acid molecule, forexample, a viral genome, a plasmid, a bacmid, a cosmid, a BAC, a YAC, achromosome of the host cell into which the nucleic acid molecule of theinvention is inserted, or any other nucleic acid molecule known to thoseskilled in the art. In some embodiments, one or more sequences encodingsuppressor tRNAs may be provided on a nucleic acid molecule comprisingall or a portion of a viral genome. In some embodiments, more than onecopy (e.g., two, three, four, five, seven, ten, twelve, fifteen, twenty,thirty, fifty, etc. copies) of the gene encoding the suppressor tRNA maybe provided. In some embodiments, the transcription of the suppressortRNA may be under the control of a regulatable (e.g., inducible orrepressible) promoter. In other embodiments, the transcription of thesuppressor tRNA may be under the control of a constitutive promoter.When more than one gene encoding a suppressor tRNA is provided, thegenes may be the same or different and may be expressed from the same ordifferent promoters.

[0048] The sequence of interest may comprise a ORF of interest that maybe provided with translation initiation signals (e.g., Shine-Delgamosequences, Kozak sequences and/or IRES sequences) in order to permit theexpression of a polypeptide from the ORF with a native N-terminus whenthe stop codon is not suppressed. Further, the sequence of interest maybe constructed by recombinational cloning of two or more differentsequences resulting in recombination sites within the sequence ofinterest. Recombination sites that reside between nucleic acid segmentsthat encode components of fusion proteins may be designed either to notencode stop codons or to not encode stop codons in the fusion proteinreading frame. A sequence of interest encoding a polypeptide may also beprovided with a stop codon (e.g., a suppressible stop codon) at the 3′end of the coding sequence. Similarly, when a fusion protein is producedfrom multiple nucleic acid segments (e.g., three, four, five, six,eight, ten, etc. segments), nucleic acids sequences that encode stopcodons can be omitted between each nucleic acid segment and/or nucleicacids that encodes a stop codon can be positioned at the 3′ end of oneor more of the segments and/or at the 3′ end of the 3′-most segment ofthe fusion protein coding region.

[0049] In some embodiments, a tag sequence may be provided at both theN- and C-termini of the gene of interest. Optionally, the tag sequenceat the N-terminus may be provided with a stop codon and an ORF ofinterest may be provided with a stop codon and the tag at the C-terminusmay be provided with a stop codon. The stop codons may be the same ordifferent.

[0050] In some embodiments, the stop codon of the N-terminal tag isdifferent from the stop codon of the ORF of interest. In embodiments ofthis type, suppressor tRNAs corresponding to one or both of the stopcodons may be provided. When both are provided, each of the suppressortRNAs may be independently provided on the same vector (e.g., plasmid,virus, etc.), on a different viral vector or other vector, or in thehost cell genome. The suppressor tRNAs need not both be provided in thesame way, for example, one may be provided on the vector contain thegene of interest while the other may be provided in the host cellgenome.

[0051] Depending on the location of the expression signals (e.g.,promoters), suppression of the stop codon(s) during expression allowsproduction of a fusion peptide having the tag sequence at the N- and/orC-terminus of the expressed protein. By not suppressing the stopcodon(s), expression of the sequence of interest without the N- and/orC-terminal tag sequence may be accomplished. Thus, the invention allowsthrough recombination efficient construction of vectors (e.g., viralvectors) containing one or more ORFs (e.g., one, two, three, four, five,six, ten, or more ORFs) or other sequence of interest (e.g.,untranslated sequences such as RNAi, tRNAs, ribozymes, etc.) forcontrolled expression of fusion proteins depending on the need. Thoseskilled in the art will appreciate that suppression is not 100%effective. Thus, under suppressing conditions a mixture of polypeptidesis produced, the mixture comprising polypeptides that terminate at thestop codon and polypeptides that contain amino acid sequences encodedafter the stop codon. For example, in the case discussed above wherethree coding regions are separated by two stop codons, under conditionsdesigned to suppress both stop codons, a mixture containing variousamounts of the polypeptide encoded by the first coding region plus apolypeptide encoded by the first and the second coding regions and apolypeptide containing amino acids of all three coding regions might beproduced.

[0052] The present invention provides methods of making stable celllines and cell lines made by the methods of the invention. Stable celllines may incorporate one or more sequences of interest that may beincorporated into the genome of the cell or may be maintainedextra-chromasomally. Optionally, a sequence of interest may include oneor more stop codons, one or more of which may be located at or near the3′ end of a coding sequence present in the sequence of interest. Astable cell line of the invention may be contacted with one or morenucleic acid molecules comprising all or a portion of a viral genomeunder conditions causing suppression of one or more of the stop codonspresent in the sequence of interest. A nucleic acid molecule comprisingall or a portion of a viral genome may also comprise one or more copies(e.g., two, three, four, five, six, seven, eight, nine, ten, fifteen,twenty, twenty five, etc.) of a sequence that produces a suppressortRNA. In the absence of the nucleic acid molecule expressing asuppressor tRNA, for example, a nucleic acid molecule comprising all ora portion of a viral genome and comprising one or more sequence encodinga suppressor tRNA, a stable cell line of the invention may express apolypeptide encoded by a sequence of interest such that the polypeptidehas a native primary structure. In the presence of a suppressorexpressing nucleic acid molecule, for example, a nucleic acid moleculecomprising all or a portion of a viral genome and comprising one or moresequence encoding a suppressor tRNA, a stable cell line of the inventionmay express a fusion protein incorporating the polypeptide encoded bythe sequence of interest and some additional peptide sequence. A stablecell line of the invention may also comprise a suppressor tRNA encodingsequence in the genome of the cell, which sequence may be under thecontrol of a promoter that is inducible (e.g., inducible by a nucleicacid molecule comprising all or a portion of a viral genome or apolypeptide encoded by such a nucleic acid molecule). Thus, contactingthe cell with a nucleic acid molecule comprising all or a portion of aviral genome may result in production of a suppressor tRNA andsuppression of one or more stop codons present in a sequence ofinterest.

[0053] The sequences of interest to be incorporated in the viral vectorsand/or nucleic acids molecules of the invention may comprise at leastone open reading frame (ORF) (e.g., one, two, three, four, five, seven,ten, twelve, or fifteen ORFs). Such sequences may also comprisefunctional sequences (e.g., primer binding sites, transcriptional ortranslation sites or signals), termination sites (e.g., stop codons thatmay be optionally suppressed), origins of replication, and the like, andoften will comprise sequences that regulate gene expression includingtranscriptional regulatory sequences and sequences that function asinternal ribosome entry sites (IRES). Often, either the sequence ofinterest and/or the portions of the nucleic acid comprising the viralgenome adjacent to the sequence of interest comprise sequences thatfunction as a promoter. Either or both the sequence of interest and/ornucleic acid comprising all or a part of a viral genome may alsocomprise transcription termination sequences, selectable markers,restriction enzyme recognition sites, and the like.

[0054] In some embodiments, nucleic acid molecules of the inventioncomprising all or a portion of a viral genome may comprise two copies ofthe same selectable marker, each copy flanked by two recombinationsites. In other embodiments, these molecules may comprise two differentselectable markers each flanked by two recombination sites. In someembodiments, one or more of these selectable markers may be a negativeselectable marker (e.g., ccdB, kicB, Herpes simplex thymidine kinase,cytosine deaminase, etc.).

[0055] In one aspect, the present invention provides a compositioncomprising a recombinant viral vector which encodes one or moresuppressor tRNAs. Such compositions may comprise any number ofadditional components, for example, cells, media, buffers, proteins,lipids, and the like. In some embodiments, the viral vector may be anadenovirus. A viral vector may encode one or more suppressor tRNAs thatrecognize one of the stop codons selected from TAG, TGA, or TAA. In someembodiments, the viral vector encodes a plurality of suppressor tRNAs,for example, eight suppressor tRNAs that recognize the stop codon TAG.

[0056] In some embodiments, the present invention provides compositionscomprising a nucleic acid molecule comprising all or a portion of atleast one viral genome and further comprising at least two recombinationsites that do not substantially recombine with each other ; and apolypeptide. Any polypeptide may be included in compositions of thistype, for example, the polypeptide may be a viral envelop polypeptide. Acomposition of this type may be in the form of a particle comprising thenucleic acid molecule and the polypeptide. All or a portion of any viralgenome may be included on the nucleic acid molecule, for example, theviral genomes may be a lentiviral genome, for example an HIV genome(such as HIV-1). A polypeptide suitable for compositions of this type isvesicular stomatitis virus G-protein.

[0057] In another aspect, the present invention provides host cellscomprising a first nucleic acid sequence encoding a fusion polypeptide,wherein the sequence comprises at least a first coding region, and asecond coding region separated by a sequence comprising a stop codon,and a second nucleic acid sequence comprising one or more suppressortRNAs that suppresses the stop codon. In some embodiments, at least oneof the first and/or second nucleic acid sequence is present on a nucleicacid molecule comprising all or a portion of at least one viral genome(e.g., an adenoviral genome). In some embodiments, the one or moresuppressor tRNAs are expressed from a nucleic acid molecule comprisingall or a portion of at least one viral genome (e.g., an adenoviralgenome). A nucleic acid molecule may encode one or more suppressor tRNAsthat recognizes one of the stop codons selected from TAG, TGA, or TAA.In some embodiments, the nucleic acid molecule may encode a plurality ofsuppressor tRNAs. In some embodiments, the nucleic acid molecule mayencode eight suppressor tRNAs that recognize the stop codon TAG and maycomprise all or a portion of an adenoviral genome.

[0058] In one aspect, the present invention provides a host cellcomprising a nucleic acid molecule comprising all or a portion of atleast one viral genome and further comprising at least two recombinationsites that do not substantially recombine with each other. In someembodiments, at least one of the viral genomes may be a lentiviralgenome (e.g., an HIV genome). In some aspects, a nucleic acid moleculemay be stably integrated into the genome of the host cell. In someembodiments, at least one of the viral genomes may be an RNA virusgenome (e.g., of the family Togaviridae or Flaviviridae such as analphavirus, a Sindbis virus and a Kunjin virus).

[0059] In one aspect, the present invention provides a method ofexpressing a polypeptide. Such methods may comprise contacting a cellwith a nucleic acid molecule comprising a sequence encoding thepolypeptide operably linked to a promoter and a repressor sequence,wherein the nucleic acid molecule comprises all or a portion of a viralgenome, contacting the cell with a nucleic acid molecule encoding aprotein that binds to the repressor sequence; and incubating the cellunder conditions sufficient to express the polypeptide. In embodimentsof this type, the viral genome may be a lentiviral genome (e.g., anHIV). In some aspects, the repressor sequence may be the tetracyclineoperator sequence and the protein may be the tetracycline repressorprotein and conditions sufficient to express the polypeptide compriseincubating the cell in the presence of a compound that reduces bindingof the protein to the repressor sequence (e.g., tetracycline).

[0060] In another aspect, the present invention provides a method ofexpressing a polypeptide, comprising contacting a cell with a nucleicacid molecule comprising a sequence encoding the polypeptide operablylinked to a promoter and a repressor sequence, wherein the nucleic acidmolecule comprises all or a portion of a viral genome and wherein thecell express a protein that binds to the repressor sequence; andincubating the cell under conditions sufficient to express thepolypeptide. In embodiments of this type, the viral genome may be alentiviral genome (e.g., an HIV). In some aspects, the repressorsequence may be the tetracycline operator sequence and the protein maybe the tetracycline repressor protein and conditions sufficient toexpress the polypeptide comprise incubating the cell in the presence ofa compound that reduces binding of the protein to the repressor sequence(e.g., tetracycline).

[0061] The present invention also relates to kits for carrying outmethods of the invention, and particularly for use in creatingrecombinant viral vectors and/or nucleic acids molecules of theinvention. Kits of the invention may also comprise further componentsfor further manipulating nucleic acids and/or viral vectors produced bymethods of the invention. Kits of the invention may comprise one or morenucleic acid molecules comprising all or a portion of a viral genome.Such kits may optionally comprise one or more additional componentsselected from the group consisting of one or more host cells (e.g., two,three, four, five etc.), one or more reagents for introducing (e.g., bytransfection or transformation) molecules or compounds into one or morehost cells, one or more nucleotides, one or more polymerases and/orreverse transcriptases (e.g., two, three, four, five, etc.), one or moresuitable buffers (e.g., two, three, four, five, etc.), one or moreprimers (e.g., two, three, four, five, seven, ten, twelve, fifteen,twenty, thirty, fifty, etc.), one or more populations of molecules forcreating combinatorial libraries (e.g., two, three, four, five, seven,ten, twelve, fifteen, twenty, thirty, fifty, etc.) and one or morecombinatorial libraries (e.g., two, three, four, five, seven, ten,twelve, fifteen, twenty, thirty, fifty, etc.). Kits of the invention mayalso contain directions or protocols for carrying out one or moremethods of the invention.

[0062] In another aspect the invention provides kits for joining,deleting, or replacing nucleic acid segments in the viral vectors and/ornucleic acids molecules of the invention, these kits comprising at leastone component selected from the group consisting of (1) one or morerecombination proteins; (2) one or more compositions comprising one ormore recombination proteins; (3) at least one nucleic acid moleculecomprising one or more recombination sites (preferably a vector havingat least two different recombination specificities); (4) one or morenucleic acid molecules comprising all or a portion of a viral genome andone or more recombination sites; (5) one or more enzymes having ligaseactivity; (6) one or more enzymes having polymerase activity; (7) one ormore enzymes having reverse transcriptase activity; (9) one or moreenzymes having restriction endonuclease activity; (10) one or moreprimers; (11) one or more nucleic acid libraries; (12) one or morereagents for introducing macromolecules into cells; (13) one or morebuffers; (14) one or more detergents or solutions containing detergents;(15) one or more nucleotides; (16) one or more terminating agents; (17)one or more transfection reagents; (18) one or more host cells; (19) oneor more topoisomerases; (20) one or more nucleic acid molecules to whichat least one topoisomerases is bound; (21) one or more nucleic acidmolecules comprising at least one topoisomerases recognition sequence;and (22) instructions for using kit components.

[0063] Further, kits of the invention may contain one or morerecombination proteins. Any recombination protein known to those skilledin the art may be provided in the kits of the invention. Examples ofsuitable recombination proteins include, but are not limited to, Cre,Int, IHF, Xis, Flp, Fis, Hin, Gin, Cin, Tn3 resolvase, ΦC31, TndX, XerC,and XerD.

[0064] In addition, kits of the invention may contain one or morenucleic acids having more than one recombination site (e.g., one or morerecombination sites with different recombination specificities such asatt sites with different seven base pair overlap regions). In specificembodiments, kits of the invention contain compositions comprising oneor more recombination proteins capable of catalyzing recombinationbetween recombination sites, e.g., between att sites. In relatedembodiments, these compositions comprise one or more recombinationproteins capable of catalyzing attB×attP (BP) reactions, attL×attR (LR)reactions, or both BP and LR reactions.

[0065] The invention also relates to compositions for carrying outmethods of the invention and to compositions created while carrying outmethods of the invention. In particular, the invention includesrecombinant viral vectors prepared by methods of the invention, methodsfor preparing host cells that contain these viral vectors, host cellsprepared by these methods, and methods employing these host cells forproducing products (e.g., RNA, protein, etc.) encoded by these viralvectors, and products encoded by these viral vectors (e.g., RNA,protein, etc.).

[0066] Compositions, methods and kits of the invention may be preparedand carried out using a phage-lambda site-specific recombination system,such as with the GATEWAY™ Recombinational Cloning System available fromInvitrogen Corporation, Carlsbad, Calif. The GATEWAY™ TechnologyInstruction Manual (catalog number 12539-011, version C, InvitrogenCorporation, Carlsbad, Calif.) describes in more detail this system andis incorporated herein by reference in its entirety.

[0067] Other embodiments of the invention will be apparent to one orordinary skill in the art in light of what is known in the art, in lightof the following drawings and description of the invention, and in lightof the claims.

BRIEF DESCRIPTION OF THE FIGURES

[0068]FIG. 1 is a schematic representation of the basic recombinationalcloning reaction.

[0069]FIG. 2 is a schematic representation of the use of the presentinvention to clone two nucleic acid segments by performing an LRrecombination reaction.

[0070]FIGS. 3A to 3D illustrate various embodiments of compositions andmethods of the invention for generating a covalently linkeddouble-stranded recombinant nucleic acid molecule. Topoisomerase isshown as a solid circle, and is either attached to a terminus of asubstrate nucleic acid molecule or is released following a linkingreaction. As illustrated, the substrate nucleic acid molecules have 5′overhangs, although they similarly can have 3′ overhangs or can be bluntended. In addition, while the illustrated nucleic acid molecules areshown having the topoisomerases bound thereto (topoisomerase-charged),one or more of the termini shown as having a topoisomerase bound theretoalso can be represented as having a topoisomerase recognition site, inwhich case the joining reaction would further require addition of one ormore site specific topoisomerases, as appropriate.

[0071]FIG. 3A shows a first nucleic acid molecule having a topoisomeraselinked to each of the 5′ terminus and 3′ terminus of one end, andfurther shows linkage of the first nucleic acid molecule to a secondnucleic acid molecule.

[0072]FIG. 3B shows a first nucleic acid molecule having a topoisomerasebound to the 3′ terminus of one end, and a second nucleic acid moleculehaving a topoisomerase bound to the 3′ terminus of one end, and furthershows a covalently linked double-stranded recombinant nucleic acidmolecule generated due to contacting the ends containing thetopoisomerase-charged substrate nucleic acid molecules.

[0073]FIG. 3C shows a first nucleic acid molecule having a topoisomerasebound to the 5′ terminus of one end, and a second nucleic acid moleculehaving a topoisomerase bound to the 5′ terminus of one end, and furthershows a covalently linked double-stranded recombinant nucleic acidmolecule generated due to contacting the ends containing thetopoisomerase-charged substrate nucleic acid molecules.

[0074]FIG. 3D shows a nucleic acid molecule having a topoisomeraselinked to each of the 5′ terminus and 3′ terminus of both ends, andfurther shows linkage of the topoisomerase-charged nucleic acid moleculeto two nucleic acid molecules, one at each end. The topoisomerases ateach of the 5′ termini and/or at each of the 3′ termini can be the sameor different.

[0075]FIG. 4 is a schematic representation of one embodiment of theinvention.

[0076]FIGS. 5A-5F are schematic representation of exemplary vectors ofthe invention. FIG. 5A depicts a vector that contains two different DNAinserts, the transcription of which is driven in different directions bypromoters (e.g., polyhedrin, p10, T7, CMV, MMTV, metalothionine, RSV,SV40, hGH promoters). Depending on the type of transcripts which are tobe produced, either of DNA-A and/or DNA-B may be in an orientation whichresults in the production of either sense or anti-sense RNA.

[0077]FIG. 5B is a schematic representation of an exemplary vector ofthe invention which contains one DNA insert, the transcription of whichmay proceed in either direction (or both directions) driven by twopromoters which may be the same or different. Thus, RNA produced bytranscription driven by one promoter will be sense RNA and RNA producedby transcription driven by the other promoter will be anti-sense RNA.RNA can be produced from both promoters, for example, to make smallinterfering RNA (siRNA).

[0078]FIG. 5C is a schematic representation of an exemplary vector ofthe invention which contains two different DNA inserts having the samenucleotide sequence (i.e., DNA-A), the transcription of which are drivenin different directions by two separate promoters, which may be the sameor different. In this example, RNA produced by transcription driven byone promoter will be sense RNA and RNA produced by transcription drivenby the other promoter will be anti-sense RNA.

[0079]FIG. 5D is a schematic representation of an exemplary vector ofthe invention that contains two DNA inserts having the same nucleotidesequence (i.e., DNA-A) in opposite orientations, the transcription ofwhich is driven by one promoter (e.g., CMV promoter). A transcriptiontermination signal is not present between the two copies of DNA-A andthe DNA-A inserts. Transcription of one segment produces a sense RNA andof the other produces an anti-sense RNA. The RNA produced from thisvector will undergo intramolecular hybridization and, thus, will form adouble-stranded molecule with a hairpin turn.

[0080]FIGS. 5E and 5F are schematic representations of two exemplaryvectors of the invention, each of which contains a DNA insert having thesame nucleotide sequence (i.e., DNA-A). Transcription of these insertsresults in the production of sense and anti-sense RNA which may thenhybridize to form double stranded RNA molecules.

[0081]FIG. 6 is a plasmid map of pAd/CMV/V5-DEST.

[0082]FIG. 7 is a plasmid map of pAd-GW-TO/tRNA.

[0083]FIG. 8 is a plasmid map of pAdenoTAG tRNA.

[0084]FIG. 9 is a plasmid map of pAd/PL-DEST.

[0085]FIG. 10 is a plasmid map of pAd/CMV/V5-GW/lacZ.

[0086]FIG. 11 shows the recombination region of pAd/CMV/V5-DEST.

[0087]FIG. 12 shows the recombination region of pAd/PL-DEST.

[0088]FIG. 13 shows a schematic representation of producing an exemplaryadenoviral vector produced as described in Example 4.

[0089] FIGS. 14A-C show the cytopathic effect (CPE) in 293A cellstransfected with Pac I-digested pAd/CMV/V5-GW/lacZ plasmid as describedin Example 4. FIG. 14A shows 293A cells at days 4-6 post-transfection.At this early stage, cells producing adenovirus first appear as patchesof rounding, dying cells. FIG. 14B shows 293A cells at day 6-8post-transfection. As the infection proceeds, cells containing viralparticles lyse and infect neighboring cells. A plaque begins to form.FIG. 14C shows cells at day 8-10 post-transfection At this late stage,infected neighboring cells lyse, forming a plaque that is clearlyvisible.

[0090]FIG. 15 is a plasmid map of pIB/V5-His-DEST.

[0091]FIG. 16 provides the nucleotide sequence of the OpIE2 promoter.

[0092]FIG. 17 shows the recombination region of pIB/V5-His-DEST.

[0093]FIG. 18 is a plasmid map of pIB/V5-His-GW/lacZ.

[0094]FIG. 19A shows a schematic representation of the BaculoDirect™V5-His Dest cassette. FIG. 19B shows a schematic representation of theBaculoDirect™ Mel/V5-His Dest cassette.

[0095]FIG. 20 shows a schematic representation of the genome of abaculovirus of the invention and an entry clone to introduce a gene ofinterest into the baculoviral genome.

[0096]FIG. 21 shows a schematic representation of the topoisomerasemediate insertion of the gp64 promoter into pIB/V5-His.

[0097]FIG. 22 is a plasmid map of pIB/V5-His/gp64/DEST.

[0098]FIG. 23 is a bar graph showing the results of a transienttransfection assay.

[0099]FIG. 24 is a Western blot showing protein expression levels ofstably transfected cells and transiently transfected cells.

[0100]FIGS. 25A and 25B are Western blots showing protein expressionlevels of stably transfected cells.

[0101]FIG. 26 is a bar graph showing the results of a lacZ transfectionassay.

[0102]FIG. 27A shows a schematic representation of the construction ofBaculoDirect™ vector. FIG. 27B shows a schematic representation of an LRreaction between the BaculoDirect™ vector and an entry clone containinga gene of interest.

[0103]FIG. 28 shows a schematic representation of a high throughputcloning protocol using the baculoviruses of the present invention.

[0104]FIG. 29 shows the results of a comparison of the use of circularvirus DNA and linear virus DNA in the initial LR clonase reaction.

[0105]FIG. 30 shows the results obtained in the presence of ganciclovirselection.

[0106]FIG. 31 shows the results of a Western blot of variouspolypeptides expressed using BaculoDirect™.

[0107]FIG. 32 shows a comparison of the titers of recombinantbaculoviruses obtained using various techniques. Virus titer wasobtained using the TCID₅₀ technique (upper panel) and by plaque assay(lower panel).

[0108]FIG. 33 shows a comparison of the cumulative time required toprepare a viral stock using Bac to Bac™ and BaculoDirect™.

[0109]FIG. 34 shows a schematic representation of plasmid pVL1393 GSTp10 stop.

[0110]FIG. 35 shows a schematic representation of a method of making anucleic acid molecule comprising all or a portion of a lentiviralgenome.

[0111]FIG. 36 shows a schematic representation of plasmids for use inthe present invention. FIG. 36A shows a schematic representationpLenti6/V5-DEST. FIG. 36B shows a schematic representation ofpLenti6/V5-D-TOPO®. FIG. 36C shows a plasmid map of pLenti4/V5-DEST.FIG. 36D shows a plasmid map of pLenti6/UbC/V5-DEST.

[0112]FIG. 37 shows a schematic representation of plasmids for use inthe present invention. FIG. 37A shows a schematic representation pLP1.FIG. 37B shows a schematic representation of pLP2. FIG. 37C shows aschematic representation of pLP/VSVG.

[0113]FIG. 38 shows the results of an experiment in which two LRreactions were performed with either pLenti6/V5-DEST alone orpLenti6/V5-DEST plus pENTR/CAT and 3 μl of each was transformed intoTOP10 cells. 100 μl of the transformations were plated on regular LB-ampplates (no Bsd) or LB-amp containing 50 μg/ml blasticidin. FIG. 38A isphotograph shown the observed colony morphologies. FIG. 38B shows theresults in tabular form.

[0114]FIGS. 39A and 39B show the results of a Western blot withanti-lacZ antibody (FIG. 39A) and anti-V5-antibody (FIG. 39B).

[0115]FIG. 40 shows in tabular form the titers of lentiviral stocksprepared with inserts of varying size.

[0116]FIGS. 41A, 41B, and 41C show the expression of marker genes usingthe lentiviral expression system. FIG. 41A shows the expression of lacZusing the GATEWAY™ adapted lentiviral system. FIGS. 41B and 41C show theexpression of GFP using the topoisomerase adapted lentiviral system.

[0117]FIGS. 42A and 42B show Western blots of the expression of variousgenes using the lentiviral expression system described herein. FIG. 42Ashows the expression of lacZ, CAT and GFP. FIG. 42B shows the expressiono PKC and GFP.

[0118]FIGS. 43A and 43B show the results of varying the multiplicity ofinfection on the observed expression level of lacZ using the lentiviralexpression system of the invention. FIG. 43A shows photographs cellsstained to detect β-galactosidase activity. FIG. 43B is a graph ofβ-galactosidase activity as a function of MOI.

[0119]FIGS. 44A and 44B show the results of transduction of various celltypes with lentiviral vectors prepared according to the methods of theinvention. FIG. 44A is a bar graph of β-galactosidase activity observedin various actively growing or G1/S arrested cell types. FIG. 44Bprovides photographs of contacted-inhibited primary foreskin cellstransduced with lentiviral vectors and stained to detect lacZ activity.

[0120]FIGS. 45A and 45B show long term expression of genes from cellstransduced with the nucleic acid molecules of the invention. FIG. 45Ashows photographs of transduced cells stained for β-galactosidaseactivity after 10 days. FIG. 45B shows photographs of transduced cellsstained for β-galactosidase activity after 6 weeks.

[0121]FIG. 46A shows the recombination region of pLenti6/V5-DEST.

[0122]FIG. 46B shows the recombination region of the expression cloneresulting from pLenti6/UbC/V5-DEST×entry clone. FIG. 46C shows thecomplete sequence of the UbC promoter.

[0123]FIG. 47 is a schematic representation of directional topoisomerasecloning according to the invention.

[0124]FIG. 48 shows the cloning region of pLenti6/V5-D-TOPO®.

[0125]FIG. 49 shows a plasmid map of pCMVSPORT6TAg.neo.

[0126]FIG. 50 shows a schematic representation of the Tag-On-Demand™method described in Example 14. A coding sequence of interest (GOI) iscloned with a TAG stop codon into an expression vector such that it isoperably linked to a promoter (as an example, the CMV promoter isindicated in the figure). If its native stop codon is not TAG, it mustbe changed to TAG to be compatible with this particular method althoughby changing the anticodon on the suppressor tRNA molecule any stop codoncan be used. Downstream of, and in frame with, the GOI is an epitope tagto be fused to the C-terminus of the protein of interest (e.g., V5, GFP,etc.). Under normal expression conditions (i.e., in the absence of tRNAsuppressor (−tRNA^(TAG))), native protein is expressed. In the presenceof the tRNA suppressor (+tRNA), the TAG stop codon is translated as aserine in this example, and translation continues along to produce atagged protein. The expression vector contains at least one non-TAG stopcodon (e.g., TAA or TGA) downstream of the C-terminal epitope tag toterminate translation of the fusion protein.

[0127] FIGS. 51A-B shows western blots from plasmid tRNA suppressionusing the V5 epitope and GFP Tag-On-Demand™ method described in Example14. FIG. 51A shows the western blots of CHO cells that wereco-transfected with one of three reporters: pcDNA3.2/V5-GW/CAT^(TAA),-GW/CAT^(TAG) or -GW/CAT^(TGA) in the presence or absence of its cognatetRNA suppressor: pUC12-tRNA^(TAA), pUC12-tRNA^(TAG) or pUC12-tRNA^(TAA),as indicated. Forty-eight hours post transfection, 20 μg of cell lysatewas analyzed by either anti-V5 or anti-CAT western blotting asindicated. A control transfection of pcDNA3.1/CAT was also included ineach experiment (CAT lane). FIG. 5 B is the western blot of 293FT cellsthat were co-transfected with one of three reporters:pcDNA6.2/GFP-GW/CAT^(TAA), -GW/CAT^(TAG) or -GW/CAT^(TGA) and one of thetRNA suppressors: pUCl2-tRNA ^(TAA), pUC12-tRNA^(TAG) orpUC12-tRNA^(TGA), as indicated. Forty-eight hours post transfection, 20μg of cell lysate was analyzed by anti-CAT western blotting asindicated. A control transfection of pcDNA3.1/CAT was also included ineach experiment (CAT lane).

[0128]FIG. 52 shows the stop codon specificity of tRNA suppression usingplasmid tRNA suppression. CHO cells were co-transfected withpcDNA3.1/lacZ-stop^(TAG)-GFP and one of each of the three tRNAsuppressors: pUC12-tRNA^(TAA), pUC12-tRNA ^(TGA) and pUC12-tRNA^(TGA).Forty-eight hours post-transfection, brightfield (upper panes) andfluorescent (lower panels) photographs were taken.

[0129]FIG. 53 shows the expression of the gene of interest afteradenovirus delivery of the monomer vs. octamer tRNA^(TAG) construct.COS-7 cells were transduced with crude lysates of Adeno-tRNA^(TAG)(monomer) or Adeno-tRNA8^(TAG) (octamer) at an MOI of 50 for 6 hours,followed by an overnight transfection with pcDNA3.1/lacZ-stop^(TAG)-GFP.72 hours post-transduction, fluorescent photographs (upper panels) andanti-lacZ western blotting (lower panel) were performed. Lane 1: mock,Lane 2: co-transfection of pUC 12-tRNA^(TAG) and reporter vector(positive control), Lane 3: Adeno-tRNA^(TAG) (monomer), Lane 4:Adeno-tRNA8^(TAG) (octamer).

[0130]FIG. 54 shows the expression of the indicated pENTR-ORF clone.

[0131] Three pENTR-ORF clones were taken from the InvitrogenCorporation, Carlsbad, Calif. human ORF collection and L×R crossed intoeither pcDNA6.2/GFP-DEST or pcDNA6.2/V5-DEST to create expressionvectors. COS-7 cells were transduced with Ad-tRNA8^(TAG) (MOI 50)followed by transfection with the ORF expression vectors. Twenty-fourhours post transfection, fluorescent photographs were taken (upperpanels). V5-western blotting was performed on RIPA lysates followingco-transfection of COS-7 cells with the ORF expression clone and thepUC12-tRNA^(TAG) (lower panel). ORF6 expresses a protein similar toCGI-130, ORF7 expresses a splicing factor and ORF12 expresses atruncated c-myc p64 protein. “lacZ” refers topcDNA3.1/lacZ-stop^(TAG)-V5 and “GFP-V5” refers to constitutive GFPexpression from pcDNA5/GFP.

[0132]FIGS. 55A and 55B shows western blots from cells transduced withadenovirus-tRNA^(TAG) for the suppression of either transient or stabletarget genes. FIG. 55A shows a western blot of the tRNA suppression of astably-expressed target gene. FlpIn-CHO cells stably expressing a singlecopy of pcDNA6/FRT/lacZ-stop-^(TAG)-GFP were transduced withAdeno-tRNA8^(TAG) at various MOIs. 48 hours post-transduction, celllysates were analyzed by anti-lacZ western blotting and percentsuppression was determined by densitometry. The additional band presentin the “stable GOI” western blot (indicated by *) is the endogenouslacZeo fusion protein present in the Flp-In CHO cell line. FIG. 55Bshows a western blot of the tRNA suppression of a transiently-expressedtarget gene. COS-7 cells were transiently transfected with the plasmidpcDNA3.1 /lacZ-stop^(TAG)-GFP following transduction with CsCl purifiedAdeno-tRNA8^(TAG) at various MOIs. 48 hours post-transduction, celllysates were analyzed by anti-lacZ western blotting and percentsuppression was determined by densitometry.

[0133]FIG. 56 shows the use of the Tag-On-Demand™ method in fivemammalian cell lines. BHK-21, CHO—S, COS-7, HeLa and HT1080 cells weretransduced with CsCl purified Adeno-tRNA8^(TAG) at an MOI of 50 followedby a transfection with pcDNA3.1/lacZ-stop^(TAG)-GFP. Brightfield (upperpanels) and fluorescent (lower panels) photographs were taken 48 hourspost transduction.

[0134]FIG. 57 is a plasmid map of pcDNA™6.2/V5-DEST.

[0135]FIG. 58 is a plasmid map of pcDNA™6.2/GFP-DEST.

[0136]FIG. 59 is a plasmid map of pcDNA™6.2/V5-GW/p64^(TAG).

[0137]FIG. 60 is a plasmid map of pcDNA™6.2/GFP-GW/p64^(TAG).

[0138]FIGS. 61A and 61B provide the sequences of the recombinationregions of vectors pcDNA™6.2/V5-DEST and pcDNA™6.2/GFP-DEST,respectively.

[0139]FIG. 62 provides a schematic representation of a method of usingan adenovirus of the invention to produce C-terminal fusion proteins ina transient transfection experiment.

[0140]FIG. 63 provides a schematic representation of a method of usingan adenovirus of the invention to produce C-terminal fusion proteins ina stable cell line containing an expression construct.

[0141]FIG. 64 shows fluorescent micrographs of GFP-fusion proteins madeusing the present invention.

[0142]FIG. 65 shows a schematic of the use of a fluorogenic substrate toassay β-lactamase activity according to one aspect of the invention.

[0143]FIG. 66 shows a comparison of sequential (left column) versussimultaneous (right column) transduction/transfection.

[0144]FIG. 67 shows Western blots showing the effects of variouslipid/DNA ratios and MOI in a simultaneous transduction/transfectionmethod (upper panels) and a sequential transduction/transfection method(lower panels).

[0145]FIG. 68 is a Western blot showing the results of an experiment inwhich COS-7 cells were transduced with an adenovirus expressingsuppressor tRNA molecules at various MOIs and simultaneously transfectedwith the pcDNA™6.2/GFP-GW/p64^(TAG) plasmid.

[0146]FIG. 69 is a vector map of pLenti6/TR, a nucleic acid molecule ofthe invention that can be used to generate blasticidin resistantmammalian cells that stably express the tetracycline repressor, TetR.

[0147]FIG. 70 is a vector map of pLenti4/TO/V5-DEST, a nucleic acidmolecule of the invention.

[0148]FIG. 71 is a vector map of pLenti6/V5.

[0149]FIG. 72 is a vector map of pLenti3/V5-TREx.

[0150]FIG. 73 shows a schematic representation of a method of attachinga topoisomerase to a nucleic acid molecule of the invention.

DETAILED DESCRIPTION OF THE INVENTION

[0151] Definitions

[0152] In the description that follows, a number of terms used inrecombinant nucleic acid technology are utilized extensively. In orderto provide a clear and more consistent understanding of thespecification and claims, including the scope to be given such terms,the following definitions are provided.

[0153] Gene: As used herein, the term “gene” refers to a nucleic acidthat contains information necessary for expression of a polypeptide,protein, or untranslated RNA (e.g., rRNA, tRNA, anti-sense RNA). Whenthe gene encodes a protein, it includes the promoter and the structuralgene open reading frame sequence (ORF), as well as other sequencesinvolved in expression of the protein. When the gene encodes anuntranslated RNA, it includes the promoter and the nucleic acid thatencodes the untranslated RNA.

[0154] Structural Gene: As used herein, the phrase “structural gene”refers to refers to a nucleic acid that is transcribed into messengerRNA that is then translated into a sequence of amino acidscharacteristic of a specific polypeptide.

[0155] Host: As used herein, the term “host” refers to any prokaryoticor eukaryotic (e.g., mammalian, insect, yeast, plant, avian, animal,etc.) organism that is a recipient of a replicable expression vector,cloning vector or any nucleic acid molecule. The nucleic acid moleculemay contain, but is not limited to, a sequence of interest, atranscriptional regulatory sequence (such as a promoter, enhancer,repressor, and the like) and/or an origin of replication. As usedherein, the terms “host,” “host cell,” “recombinant host” and“recombinant host cell” may be used interchangeably. For examples ofsuch hosts, see Sambrook, et al., Molecular Cloning: A LaboratoryManual, Cold Spring Harbor Laboratory, Cold Spring Harbor, N.Y.

[0156] Transcriptional Regulatory Sequence: As used herein, the phrase“transcriptional regulatory sequence” refers to a functional stretch ofnucleotides contained on a nucleic acid molecule, in any configurationor geometry, that act to regulate the transcription of (1) one or morestructural genes (e.g., two, three, four, five, seven, ten, etc.) intomessenger RNA or (2) one or more genes into untranslated RNA. Examplesof transcriptional regulatory sequences include, but are not limited to,promoters, enhancers, repressors, operators (e.g., the tet operator),and the like.

[0157] Promoter: As used herein, a promoter is an example of atranscriptional regulatory sequence, and is specifically a nucleic acidgenerally described as the 5′-region of a gene located proximal to thestart codon or nucleic acid that encodes untranslated RNA. Thetranscription of an adjacent nucleic acid segment is initiated at ornear the promoter. A repressible promoter's rate of transcriptiondecreases in response to a repressing agent. An inducible promoter'srate of transcription increases in response to an inducing agent. Aconstitutive promoter's rate of transcription is not specificallyregulated, though it can vary under the influence of general metabolicconditions.

[0158] Target Nucleic Acid Molecule: As used herein, the phrase “targetnucleic acid molecule” refers to a nucleic acid segment of interest,preferably nucleic acid that is to be acted upon using the compounds andmethods of the present invention. Such target nucleic acid molecules maycontain one or more (e.g., two, three, four, five, seven, ten, twelve,fifteen, twenty, thirty, fifty, etc.) genes or one or more portions ofgenes.

[0159] Insert Donor: As used herein, the phrase “Insert Donor” refers toone of the two parental nucleic acid molecules (e.g., RNA or DNA) of thepresent invention that carries the an insert (see FIG. 1). The InsertDonor molecule comprises the insert flanked on both sides withrecombination sites. The Insert Donor can be linear or circular. In oneembodiment of the invention, the Insert Donor is a circular nucleic acidmolecule, optionally supercoiled, and further comprises a cloning vectorsequence outside of the recombination signals. When a population ofinserts or population of nucleic acid segments are used to make theInsert Donor, a population of Insert Donors result and may be used inaccordance with the invention. An Insert Donor may be referred to as anEntry Clone.

[0160] Insert: As used herein, the term “insert” refers to a desirednucleic acid segment that is a part of a larger nucleic acid molecule.In many instances, the insert will be introduced into the larger nucleicacid molecule. For example, the nucleic acid segments labeled ccdB,DNA-A, and DNA-B in FIG. 2, are nucleic acid inserts with respect to thelarger nucleic acid molecule shown therein. In most instances, theinsert will be flanked by recombination sites, topoisomerase sitesand/or other recognition sequences (e.g. at least one recognitionsequence will be located at each end). In certain embodiments, however,the insert will only contain a recognition sequence on one end.

[0161] Product: As used herein, the term “Product” refers to one thedesired daughter molecules comprising the A and D sequences that isproduced after the second recombination event during the recombinationalcloning process (see FIG. 1). The Product contains the nucleic acid thatwas to be cloned or subcloned. In accordance with the invention, when apopulation of Insert Donors are used, the resulting population ofProduct molecules will contain all or a portion of the population ofInserts of the Insert Donors and preferably will contain arepresentative population of the original molecules of the InsertDonors.

[0162] Byproduct: As used herein, the term “Byproduct” refers to adaughter molecule (a new clone produced after the second recombinationevent during the recombinational cloning process) lacking the segmentthat is desired to be cloned or subcloned.

[0163] Cointegrate: As used herein, the term “Cointegrate” refers to atleast one recombination intermediate nucleic acid molecule of thepresent invention that contains both parental (starting) molecules.Cointegrates may be linear or circular. RNA and polypeptides may beexpressed from cointegrates using an appropriate host cell strain, forexample E. coli DB3.1 (particularly E. coli LIBRARY EFFICIENCY® DB3.1™Competent Cells), and selecting for both selection markers found on thecointegrate molecule.

[0164] Recognition Sequence: As used herein, the phrase “recognitionsequence” or “recognition site” refers to a particular sequence to whicha protein, chemical compound, DNA, or RNA molecule (e.g., restrictionendonuclease, a modification methylase, topoisomerases, or arecombinase) recognizes and binds. In the present invention, arecognition sequence may refer to a recombination site or topoisomerasessite. For example, the recognition sequence for Cre recombinase is loxPwhich is a 34 base pair sequence comprising two 13 base pair invertedrepeats (serving as the recombinase binding sites) flanking an 8 basepair core sequence (see FIG. 1 of Sauer, B., Current Opinion inBiotechnology 5:521-527 (1994)). Other examples of recognition sequencesare the attB, attP, attL, and attR sequences, which are recognized bythe recombinase enzyme λ Integrase. attB is an approximately 25 basepair sequence containing two 9 base pair core-type Int binding sites anda 7 base pair overlap region. attP is an approximately 240 base pairsequence containing core-type Int binding sites and arm-type Int bindingsites as well as sites for auxiliary proteins integration host factor(IHF), FIS and excisionase (Xis) (see Landy, Current Opinion inBiotechnology 3:699-707 (1993)). Such sites may also be engineeredaccording to the present invention to enhance production of products inthe methods of the invention. For example, when such engineered siteslack the P1 or H1 domains to make the recombination reactionsirreversible (e.g., attR or attP), such sites may be designated attR′ orattP′ to show that the domains of these sites have been modified in someway.

[0165] Recombination Proteins: As used herein, the phrase “recombinationproteins” includes excisive or integrative proteins, enzymes, co-factorsor associated proteins that are involved in recombination reactionsinvolving one or more recombination sites (e.g., two, three, four, five,seven, ten, twelve, fifteen, twenty, thirty, fifty, etc.), which may bewild-type proteins (see Landy, Current Opinion in Biotechnology3:699-707 (1993)), or mutants, derivatives (e.g., fusion proteinscontaining the recombination protein sequences or fragments thereof),fragments, and variants thereof. Examples of recombination proteinsinclude Cre, Int, IHF, Xis, Flp, Fis, Hin, Gin, ΦC31, Cin, Tn3resolvase, TndX, XerC, XerD, TnpX, Hjc, SpCCE1, and ParA.

[0166] Recombinases: As used herein, the term “recombinases” is used torefer to the protein that catalyzes strand cleavage and re-ligation in arecombination reaction. Site-specific recombinases are proteins that arepresent in many organisms (e.g., viruses and bacteria) and have beencharacterized as having both endonuclease and ligase properties. Theserecombinases (along with associated proteins in some cases) recognizespecific sequences of bases in a nucleic acid molecule and exchange thenucleic acid segments flanking those sequences. The recombinases andassociated proteins are collectively referred to as “recombinationproteins” (see, e.g., Landy, A., Current Opinion in Biotechnology3:699-707 (1993)).

[0167] Numerous recombination systems from various organisms have beendescribed. See, e.g., Hoess, et al., Nucleic Acids Research 14(6):2287(1986); Abremski, et al., J. Biol. Chem. 261(1):391 (1986); Campbell, J.Bacteriol. 174(23):7495 (1992); Qian, et al., J. Biol. Chem.267(11):7794 (1992); Araki, et al., J. Mol. Biol. 225(1):25 (1992);Maeser and Kahnmann, Mol. Gen. Genet. 230:170-176) (1991); Esposito, etal., Nucl. Acids Res. 25(18):3605 (1997). Many of these belong to theintegrase family of recombinases (Argos, et al., EMBO J. 5:433-440(1986); Voziyanov, et al., Nucl. Acids Res. 27:930 (1999)). Perhaps thebest studied of these are the Integrase/att system from bacteriophage λ(Landy, A. Current Opinions in Genetics and Devel. 3:699-707 (1993)),the Cre/loxP system from bacteriophage P1 (Hoess and Abremski (1990) InNucleic Acids and Molecular Biology, vol. 4. Eds.: Eckstein and Lilley,Berlin-Heidelberg: Springer-Verlag; pp. 90-109), and the FLP/FRT systemfrom the Saccharomyces cerevisiae 2μ circle plasmid (Broach, et al.,Cell 29:227-234 (1982)).

[0168] Recombination Site: A used herein, the phrase “recombinationsite” refers to a recognition sequence on a nucleic acid molecule thatparticipates in an integration/recombination reaction by recombinationproteins. Recombination sites are discrete sections or segments ofnucleic acid on the participating nucleic acid molecules that arerecognized and bound by a site-specific recombination protein during theinitial stages of integration or recombination. For example, therecombination site for Cre recombinase is loxP, which is a 34 base pairsequence comprised of two 13 base pair inverted repeats (serving as therecombinase binding sites) flanking an 8 base pair core sequence (seeFIG. 1 of Sauer, B., Curr. Opin. Biotech. 5:521-527 (1994)). Otherexamples of recombination sites include the attB, attP, attL, and attRsequences described in U.S. provisional patent applications 60/136,744,filed May 28, 1999, and 60/188,000, filed Mar. 9, 2000, and inco-pending U.S. patent applications Ser. Nos. 09/517,466 and09/732,91—all of which are specifically incorporated herein byreference-and mutants, fragments, variants and derivatives thereof,which are recognized by the recombination protein λ Int and by theauxiliary proteins integration host factor (IHF), FIS and excisionase(Xis) (see Landy, Curr. Opin. Biotech. 3:699-707 (1993)).

[0169] Recombination sites may be added to molecules by any number ofknown methods. For example, recombination sites can be added to nucleicacid molecules by blunt end ligation, PCR performed with fully orpartially random primers, or inserting the nucleic acid molecules intoan vector using a restriction site flanked by recombination sites.

[0170] Topoisomerase recognition site. As used herein, the term“topoisomerase recognition site” or “topoisomerase site” means a definednucleotide sequence that is recognized and bound by a site specifictopoisomerase. For example, the nucleotide sequence 5′-(C/T)CCTT-3′ is atopoisomerase recognition site that is bound specifically by mostpoxvirus topoisomerases, including vaccinia virus DNA topoisomerase I,which then can cleave the strand after the 3′-most thymidine of therecognition site to produce a nucleotide sequence comprising5′-(C/T)CCTT-PO₄-TOPO, i.e., a complex of the topoisomerase covalentlybound to the 3′ phosphate through a tyrosine residue in thetopoisomerase (see Shuman, J. Biol. Chem. 266:11372-11379, 1991;Sekiguchi and Shuman, Nucl. Acids Res. 22:5360-5365, 1994; each of whichis incorporated herein by reference; see, also, U.S. Pat. No. 5,766,891;PCT/US95/16099; and PCT/US98/12372 also incorporated herein byreference). In comparison, the nucleotide sequence 5′-GCAACTT-3′ is thetopoisomerase recognition site for type IA E. coli topoisomerase III.

[0171] Recombinational Cloning: As used herein, the phrase“recombinational cloning” refers to a method, such as that described inU.S. Pat. Nos. 5,888,732; 6,143,557; 6,171,861; 6,270,969; and 6,277,608(the contents of which are fully incorporated herein by reference),whereby segments of nucleic acid molecules or populations of suchmolecules are exchanged, inserted, replaced, substituted or modified, invitro or in vivo. Preferably, such cloning method is an in vitro method.

[0172] Cloning systems that utilize recombination at definedrecombination sites have been previously described in U.S. Pat. No.5,888,732, U.S. Pat. No. 6,143,557, U.S. Pat. No. 6,171,861, U.S. Pat.No. 6,270,969, and U.S. Pat. No. 6,277,608, and in pending U.S.application Ser. No. 09/517,466 filed Mar. 2, 2000, and in publishedU.S. application No. 2002 0007051-A1, all assigned to the InvitrogenCorporation, Carlsbad, Calif., the disclosures of which are specificallyincorporated herein in their entirety. In brief, the GATEWAY™ CloningSystem described in these patents and applications utilizes vectors thatcontain at least one recombination site to clone desired nucleic acidmolecules in vivo or in vitro. In some embodiments, the system utilizesvectors that contain at least two different site-specific recombinationsites that may be based on the bacteriophage lambda system (e.g., att1and att2) that are mutated from the wild-type (att0) sites. Each mutatedsite has a unique specificity for its cognate partner att site (i.e.,its binding partner recombination site) of the same type (for exampleattB1 with attP1, or attL1 with attR1) and will not cross-react withrecombination sites of the other mutant type or with the wild-type att0site. Different site specificities allow directional cloning or linkageof desired molecules thus providing desired orientation of the clonedmolecules. Nucleic acid fragments flanked by recombination sites arecloned and subcloned using the GATEWAY™ system by replacing a selectablemarker (for example, ccdB) flanked by att sites on the recipient plasmidmolecule, sometimes termed the Destination Vector. Desired clones arethen selected by transformation of a ccdB sensitive host strain andpositive selection for a marker on the recipient molecule. Similarstrategies for negative selection (e.g., use of toxic genes) can be usedin other organisms such as thymidine kinase (TK) in mammals and insects.

[0173] Mutating specific residues in the core region of the att site cangenerate a large number of different att sites. As with the att1 andatt2 sites utilized in GATEWAY™, each additional mutation potentiallycreates a novel att site with unique specificity that will recombineonly with its cognate partner att site bearing the same mutation andwill not cross-react with any other mutant or wild-type att site. Novelmutated att sites (e.g., attB 1-10, attP 1-10, attR 1-10 and attL 1-10)are described in previous patent application Ser. No. 09/517,466, filedMar. 2, 2000, which is specifically incorporated herein by reference.Other recombination sites having unique specificity (i.e., a first sitewill recombine with its corresponding site and will not recombine or notsubstantially recombine with a second site having a differentspecificity) may be used to practice the present invention. Examples ofsuitable recombination sites include, but are not limited to, loxPsites; loxP site mutants, variants or derivatives such as loxP511 (seeU.S. Pat. No. 5,851,808); frt sites; frt site mutants, variants orderivatives; dif sites; dif site mutants, variants or derivatives; psisites; psi site mutants, variants or derivatives; cer sites; and cersite mutants, variants or derivatives.

[0174] Repression Cassette: As used herein, the phrase “repressioncassette” refers to a nucleic acid segment that contains a repressor ora selectable marker present in the subcloning vector.

[0175] Selectable Marker: As used herein, the phrase “selectable marker”refers to a nucleic acid segment that allows one to select for oragainst a molecule (e.g., a replicon) or a cell that contains it and/orpermits identification of a cell or organism that contains or does notcontain the nucleic acid segment. Frequently, selection and/oridentification occur under particular conditions and do not occur underother conditions.

[0176] Markers can encode an activity, such as, but not limited to,production of RNA, peptide, or protein, or can provide a binding sitefor RNA, peptides, proteins, inorganic and organic compounds orcompositions and the like. Examples of selectable markers include butare not limited to: (1) nucleic acid segments that encode products thatprovide resistance against otherwise toxic compounds (e.g.,antibiotics); (2) nucleic acid segments that encode products that areotherwise lacking in the recipient cell (e.g., tRNA genes, auxotrophicmarkers); (3) nucleic acid segments that encode products that suppressthe activity of a gene product; (4) nucleic acid segments that encodeproducts that can be readily identified (e.g., phenotypic markers suchas β-lactamase, β-galactosidase, green fluorescent protein (GFP), yellowflourescent protein (YFP), red fluorescent protein (RFP), cyanfluorescent protein (CFP), and cell surface proteins); (5) nucleic acidsegments that bind products that are otherwise detrimental to cellsurvival and/or function; (6) nucleic acid segments that otherwiseinhibit the activity of any of the nucleic acid segments described inNos. 1-5 above (e.g., antisense oligonucleotides); (7) nucleic acidsegments that bind products that modify a substrate (e.g., restrictionendonucleases); (8) nucleic acid segments that can be used to isolate oridentify a desired molecule (e.g., specific protein binding sites); (9)nucleic acid segments that encode a specific nucleotide sequence thatcan be otherwise non-functional (e.g., for PCR amplification ofsubpopulations of molecules); (10) nucleic acid segments that, whenabsent, directly or indirectly confer resistance or sensitivity toparticular compounds; and/or (11) nucleic acid segments that encodeproducts that either are toxic (e.g., Diphtheria toxin) or convert arelatively non-toxic compound to a toxic compound (e.g., Herpes simplexthymidine kinase, cytosine deaminase) in recipient cells; (12) nucleicacid segments that inhibit replication, partition or heritability ofnucleic acid molecules that contain them; and/or (13) nucleic acidsegments that encode conditional replication functions, e.g.,replication in certain hosts or host cell strains or under certainenvironmental conditions (e.g., temperature, nutritional conditions,etc.).

[0177] Selection and/or identification may be accomplished usingtechniques well known in the art. For example, a selectable marker mayconfer resistance to an otherwise toxic compound and selection may beaccomplished by contacting a population of host cells with the toxiccompound under conditions in which only those host cells containing theselectable marker are viable. In another example, a selectable markermay confer sensitivity to an otherwise benign compound and selection maybe accomplished by contacting a population of host cells with the benigncompound under conditions in which only those host cells that do notcontain the selectable marker are viable. A selectable marker may makeit possible to identify host cells containing or not containing themarker by selection of appropriate conditions. In one aspect, aselectable marker may enable visual screening of host cells to determinethe presence or absence of the marker. For example, a selectable markermay alter the color and/or fluorescence characteristics of a cellcontaining it. This alteration may occur in the presence of one or morecompounds, for example, as a result of an interaction between apolypeptide encoded by the selectable marker and the compound (e.g., anenzymatic reaction using the compound as a substrate). Such alterationsin visual characteristics can be used to physically separate the cellscontaining the selectable marker from those not contain it by, forexample, fluorescent activated cell sorting (FACS).

[0178] Multiple selectable markers may be simultaneously used todistinguish various populations of cells. For example, a nucleic acidmolecule of the invention may have multiple selectable markers, one ormore of which may be removed from the nucleic acid molecule by asuitable reaction (e.g., a recombination reaction). After the reaction,the nucleic acid molecules may be introduced into a host cell populationand those host cells comprising nucleic acid molecules having all of theselectable markers may be distinguished from host cells comprisingnucleic acid molecules in which one or more selectable markers have beenremoved (e.g., by the recombination reaction). For example, a nucleicacid molecule of the invention may have a blasticidin resistance markeroutside a pair of recombination sites and a β-lactamase encodingselectable marker inside the recombination sites. After a recombinationreaction and introduction of the reaction mixture into a cellpopulation, cells comprising any nucleic acid molecule can be selectedfor by contacting the cell population with blasticidin. Those cellcomprising a nucleic acid molecule that has undergone a recombinationreaction can be distinguished from those containing an unreacted nucleicacid molecules by contacting the cell population with a fluorogenicβ-lactamase substrate as described below and observing the fluorescenceof the cell population. Optionally, the desired cells can be physicallyseparated from undesirable cells, for example, by FACS.

[0179] In a specific embodiment of the invention, a selectable markermay be a nucleic acid sequence encoding a polypeptide having anenzymatic activity (e.g., β-lactamase activity). Assays for β-lactamaseactivity are known in the art. U.S. Pat. No. 5,955,604, issued to Tsien,et al. Sep. 21, 1999, U.S. Pat. No. 5,741,657 issued to Tsien, et al.,Apr. 21, 1998, 6,031,094, issued to Tsien, et al., Feb. 29, 2000, U.S.Pat. No. 6,291,162, issued to Tsien, et al., Sep. 18, 2001, and U.S.Pat. No. 6,472,205, issued to Tsien, et al. Oct. 29, 2002, disclose theuse of β-lactamase as a reporter gene and fluorogenic substrates for usein detecting β-lactamase activity and are specifically incorporatedherein by reference. In one embodiment of the invention, a selectablemarker may be a nucleic acid sequence encoding a polypeptide havingβ-lactamase activity and desired host cells may be identified byassaying the host cells for β-lactamase activity.

[0180] A β-lactamase catalyzes the hydrolysis of a β-lactam ring. Thoseskilled in the art will appreciate that the sequences of a number ofpolypeptides having β-lactamase activity are known. In addition to thespecific β-lactamases disclosed in the Tsien, et al. patents listedabove, any polypeptide having β-lactamase activity is suitable for usein the present invention.

[0181] β-lactamases are classified based on amino acid and nucleotidesequence (Ambler, R. P., Phil. Trans. R. Soc. Lond. [Ser.B.] 289:321-331 (1980)) into classes A-D. Class A β-lactamases possess a serinein the active site and have an approximate weight of 29 kD. This classcontains the plasmid-mediated TEM β-lactamases such as the RTEM enzymeof pBR322. Class B β-lactamases have an active-site zinc bound to acysteine residue. Class C enzymes have an active site serine and amolecular weight of approximately 39 kD, but have no amino acid homologyto the class A enzymes. Class D enzymes also contain an active siteserine. Representative examples of each class are provided below withthe accession number at which the sequence of the enzyme may be obtainedin the indicated database. Class A β-lactamases Bacteroides fragilisCS30 L13472 GenBank Bacteroides uniformis WAL-7088 P30898 SWISS-PROTPER-1, P. aeruginosa RNL-1 P37321 SWISS-PROT Bacteroides vulgatus CLA341P30899 SWISS-PROT OHIO-1, Enterobacter cloacae P18251 SWISS-PROT SHV-1,K. pneumoniae P23982 SWISS-PROT LEN-1, K. pneumoniae LEN-1 P05192SWISS-PROT TEM-1, E. coli POO810 SWISS-PROT Proteus mirabilis GN179P30897 SWISS-PROT PSE-4, P. aeruginosa Dalgleish P16897 SWISS-PROTRhodopseudomonas capsulatus SP108 P14171 SWISS-PROT NMC, E. cloacaeNOR-1 P52663 SWISS-PROT Sme-1, Serratia marcescens S6 P52682 SWISS-PROTOXY-2, Klebsiella oxytoca D488 P23954 SWISS-PROT K. oxytocaE23004/SL781/SL7811 P22391 SWISS-PROT S. typhimurium CAS-5 X92507GenBank MEN-1, E. coli MEN P28585 SWISS-PROT Serratia fonticola CUVP80545 SWISS-PROT Citrobacter diversus ULA27 P22390 SWISS-PROT Proteusvulgaris 5E78-1 P52664 SWISS-PROT Burkholderia cepacia 249 U85041GenBank Yersinia enterocolitica serotype O:3/Y-56 Q01166 SWISS-PROT M.tuberculosis H37RV Q10670 SWISS-PROT S. clavuligerusNRRL 3585 Z54190GenBank III, Bacillus cereus 569/H P06548 SWISS-PROT B. licheniformis749/C P00808 SWISS-PROT I, Bacillus mycoides NI10R P28018 SWISS-PROT I,B. cereus 569/H/9 P00809 SWISS-PROT I, B. cereus 5/B P10424 SWISS-PROTB. subtilis 168/6GM P39824 SWISS-PROT 2, Streptomyces cacaoi DSM40057P14560 SWISS-PROT Streptomyces badius DSM40139 P35391 SWISS-PROTActinomadura sp. strain R39 X53650 GenBank Nocardia lactamdurans LC411Q06316 SWISS-PROT S. cacaoi KCC S0352 Q03680 SWISS-PROT ROB-1, H.influenzae F990/LNPB51/ P33949 SWISS-PROT serotype A1 Streptomycesfradiae DSM40063 P35392 SWISS-PROT Streptomyces lavendulae DSM2014P35393 SWISS-PROT Streptomyces albus G P14559 SWISS-PROT S. lavendulaeKCCS0263 D12693 GenBank Streptomyces aureofaciens P10509 SWISS-PROTStreptomyces cellulosae KCCS0127 Q06650 SWISS-PROT Mycobacteriumfortuitum L25634 GenBank S. aureus PC1/SK456/NCTC9789 P00807 SWISS-PROTBRO-1, Moraxella catarrhalis ATCC Z54181 GenBank 53879 Q59514 SWISS-PROTClass B β-lactamase II, B. cereus 569/H P04190 SWISS-PROT II, Bacillussp. 170 P10425 SWISS-PROT II, B. cereus 5/B/6 P14488 SWISS-PROTChryseobacterium meningosepticum X96858 GenBank CCUG4310 IMP-1, S.marcescens AK9373/TN9106 P52699 SWISS-PROT B. fragilis TAL3636/TAL2480P25910 SWISS-PROT Aeromonas hydrophila AE036 P26918 SWISS-PROT L1,Xanthomonas maltophilia IID 1275 P52700 SWISS-PROT Class C β-lactamaseCitrobacter freundii OS60/GN346 P05193 SWISS-PROT E. coli K-12/MG1655P00811 SWISS-PROT P99, E. cloacae P99/Q908R/MHN1 P05364 SWISS-PROT Y.enterocolitica IP97/serotype O:5B P45460 SWISS-PROT Morganella morganiiSLM01 Y10283 GenBank A. sobria 163a X80277 GenBank FOX-3, K. oxytoca1731 Y11068 GenBank K. pneumoniae NU2936 D13304 GenBank P. aeruginosaPAO1 P24735 SWISS-PROT S. marcescens SR50 P18539 SWISS-PROTPsychrobacter immobilis A5 X83586 GenBank Class D β-lactamases OXA-18,Pseudomonas aeruginosa Mus U85514 GenBank OXA-9, Klebsiella pneumoniaeP22070 SWISS-PROT Aeromonas sobria AER 14 X80276 GenBank OXA-1,Escherichia coli K10-35 P13661 SWISS-PROT OXA-7, E. coli 7181 P35695SWISS-PROT OXA-11, P. aeruginosa ABD Q06778 SWISS-PROT OXA-5, P.aeruginosa 76072601 Q00982 SWISS-PROT LCR-1, P. aeruginosa 2293E Q00983SWISS-PROT OXA-2, Salmonella typhimurium type 1A P05191 SWISS-PROT

[0182] For additional β-lactamases and a more detailed description ofsubstrate specificities, consult Bush et al. (1995) Antimicrob. AgentsChemother. 39:1211-1233. Those skilled in the art will appreciate thatthe polypeptides having β-lactamase activity disclosed herein may bealtered by for example, mutating, deleting, and/or adding one or moreamino acids and may still be used in the practice of the invention solong as the polypeptide retains detectable β-lactamase activity. Anexample of a suitably altered polypeptide having β-lactamase activity isone from which a signal peptide sequence has been deleted and/or alteredsuch that the polypeptide is retained in the cytosol of prokaryoticand/or eukaryotic cells. The amino acid sequence of one such polypeptideis provided in Table 30.

[0183] As described in the above-referenced United States patents, hostcells to be assayed may be contacted with a fluorogenic substrate forβ-lactamase activity. In the presence of β-lactamase, the substrate iscleaved and the fluorescence emission spectrum of the substrate isaltered. As an example, un-cleaved substrate may fluoresce green (i.e.,have an emission maxima at approximately 520 nm) when excited with lighthaving a wavelength of 405 nm and the cleaved substrate may fluoresceblue (i.e., have an emission maxima at approximately 447 nm). Bydetermining the ratio of green fluorescence intensity to bluefluorescence intensity it is possible to determine the amount ofβ-lactamase produced and from that, to calculate what % of the cellsexpress β-lactamase. Kits for conducting a fluorescence-basedβ-lactamase assay are commercially available, for example, fromPanVerra, LLC, Madison, Wis., catalog number K1032.

[0184] Preferred β-lactam fluorogenic substrates for use in the presentinvention include those which comprise a fluorescence donor moiety and afluorescence acceptor moiety linked to a cephalosporin backbone suchthat, upon hydrolysis of the β-lactam, the acceptor moiety is releasedfrom the molecule. Before the β-lactam is hydrolyzed, the donor andacceptor moiety are positioned such that efficient fluorescenceresonance energy transfer (FRET) occurs. Upon excitation with light of asuitable wavelength, fluorescence from the acceptor moiety is observed.After hydrolysis of the β-lactam, the acceptor moiety is released fromthe molecule and the FRET is disrupted resulting in a change in thefluorescence emission spectrum. An example of a suitable fluorescencedonor molecule is a coumarin or derivative thereof (e.g.,6-chloro-7-hydroxycoumarin) and examples of suitable acceptor moietiesinclude, but are not limited to, fluorescein, rhodol, or rhodamine orderivatives thereof. Examples of suitable substrates include CCF2 andthe acetoxymethyl ester derivative thereof (CCF2/AM). Those skilled inthe art will appreciate that CCF2/AM is membrane permeable and isconverted to CCF2 inside a cell by the action of endogenous esteraseenzymes. A schematic showing the result of hydrolysis of CCF2 by aβ-lactamase is shown in FIG. 65.

[0185] Selection Scheme: As used herein, the phrase “selection scheme”refers to any method that allows selection, enrichment, oridentification of a desired nucleic acid molecules or host cellscontaining them (in particular Product or Product(s) from a mixturecontaining an Entry Clone or Vector, a Destination Vector, a DonorVector, an Expression Clone or Vector, any intermediates (e.g., aCointegrate or a replicon), and/or Byproducts). In one aspect, selectionschemes of the invention rely on one or more selectable markers. Theselection schemes of one embodiment have at least two components thatare either linked or unlinked during recombinational cloning. Onecomponent is a selectable marker. The other component controls theexpression in vitro or in vivo of the selectable marker, or survival ofthe cell (or the nucleic acid molecule, e.g., a replicon) harboring theplasmid carrying the selectable marker. Generally, this controllingelement will be a repressor or inducer of the selectable marker, butother means for controlling expression or activity of the selectablemarker can be used. Whether a repressor or activator is used will dependon whether the marker is for a positive or negative selection, and theexact arrangement of the various nucleic acid segments, as will bereadily apparent to those skilled in the art. In some preferredembodiments, the selection scheme results in selection of, or enrichmentfor, only one or more desired nucleic acid molecules (such as Products).As defined herein, selecting for a nucleic acid molecule includes (a)selecting or enriching for the presence of the desired nucleic acidmolecule (referred to as a “positive selection scheme”), and (b)selecting or enriching against the presence of nucleic acid moleculesthat are not the desired nucleic acid molecule (referred to as a“negative selection scheme”).

[0186] In one embodiment, the selection schemes (which can be carriedout in reverse) will take one of three forms, which will be discussed interms of FIG. 1. The first, exemplified herein with a selectable markerand a repressor therefore, selects for molecules having segment D andlacking segment C. The second selects against molecules having segment Cand for molecules having segment D. Possible embodiments of the secondform would have a nucleic acid segment carrying a gene toxic to cellsinto which the in vitro reaction products are to be introduced. A toxicgene can be a nucleic acid that is expressed as a toxic gene product (atoxic protein or RNA), or can be toxic in and of itself. (In the lattercase, the toxic gene is understood to carry its classical definition of“heritable trait.”)

[0187] Examples of such toxic gene products are well known in the art,and include, but are not limited to, restriction endonucleases (e.g.,DpnI, Nla3, etc.); apoptosis-related genes (e.g., ASK1 or members of thebcl-2/ced-9 family); retroviral genes; including those of the humanimmunodeficiency virus (HIV); defensins such as NP-1; inverted repeatsor paired palindromic nucleic acid sequences; bacteriophage lytic genessuch as those from ΦX174 or bacteriophage T4; antibiotic sensitivitygenes such as rpsL; antimicrobial sensitivity genes such as pheS;plasmid killer genes' eukaryotic transcriptional vector genes thatproduce a gene product toxic to bacteria, such as GATA-1; genes thatkill hosts in the absence of a suppressing function, e.g., kicB, ccdB,ΦX174 E (Liu, Q., et al., Curr. Biol. 8:1300-1309 (1998)); and othergenes that negatively affect replicon stability and/or replication. Atoxic gene can alternatively be selectable in vitro, e.g., a restrictionsite.

[0188] Many genes coding for restriction endonucleases operably linkedto inducible promoters are known, and may be used in the presentinvention (see, e.g., U.S. Pat. No. 4,960,707 (DpnI and DpnII); U.S.Pat. Nos. 5,082,784 and 5,192,675 (KpnI); U.S. Pat. No. 5,147,800(NgoAIII and NgoAI); U.S. Pat. No. 5,179,015 (FspI and HaeIII): U.S.Pat. No. 5,200,333 (HaeII and TaqI); U.S. Pat. No. 5,248,605 (HpaII);U.S. Pat. No. 5,312,746 (ClaI); U.S. Pat. Nos. 5,231,021 and 5,304,480(XhoI and XhoII); U.S. Pat. No. 5,334,526 (AluI); U.S. Pat. No.5,470,740 (NsiI); U.S. Pat. No. 5,534,428 (SstI/SacI); U.S. Pat. No.5,202,248 (NcoI); U.S. Pat. No. 5,139,942 (NdeI); and U.S. Pat. No.5,098,839 (Pacd). (See also Wilson, G. G., Nucl. Acids Res. 19:2539-2566(1991); and Lunnen, K. D., et al., Gene 74:25-32 (1988)).

[0189] In the second form, segment D carries a selectable marker. Thetoxic gene would eliminate transformants harboring the Vector Donor,Cointegrate, and Byproduct molecules, while the selectable marker can beused to select for cells containing the Product and against cellsharboring only the Insert Donor.

[0190] The third form selects for cells that have both segments A and Din cis on the same molecule, but not for cells that have both segmentsin trans on different molecules. This could be embodied by a selectablemarker that is split into two inactive fragments, one each on segments Aand D.

[0191] The fragments are so arranged relative to the recombination sitesthat when the segments are brought together by the recombination event,they reconstitute a functional selectable marker. For example, therecombinational event can link a promoter with a structural nucleic acidmolecule (e.g., a gene), can link two fragments of a structural nucleicacid molecule, or can link nucleic acid molecules that encode aheterodimeric gene product needed for survival, or can link portions ofa replicon.

[0192] Site-Specific Recombinase: As used herein, the phrase“site-specific recombinase” refers to a type of recombinase thattypically has at least the following four activities (or combinationsthereof): (1) recognition of specific nucleic acid sequences; (2)cleavage of said sequence or sequences; (3) topoisomerase activityinvolved in strand exchange; and (4) ligase activity to reseal thecleaved strands of nucleic acid (see Sauer, B., Current Opinions inBiotechnology 5:521-527 (1994)). Conservative site-specificrecombination is distinguished from homologous recombination andtransposition by a high degree of sequence specificity for bothpartners. The strand exchange mechanism involves the cleavage andrejoining of specific nucleic acid sequences in the absence of DNAsynthesis (Landy, A. (1989) Ann. Rev. Biochem. 58:913-949).

[0193] Suppressor tRNAs. A tRNA molecule that results in theincorporation of an amino acid in a polypeptide in a positioncorresponding to a stop codon in the mRNA being translated.

[0194] Homologous Recombination: As used herein, the phrase “homologousrecombination” refers to the process in which nucleic acid moleculeswith similar nucleotide sequences associate and exchange nucleotidestrands. A nucleotide sequence of a first nucleic acid molecule that iseffective for engaging in homologous recombination at a predefinedposition of a second nucleic acid molecule will therefore have anucleotide sequence that facilitates the exchange of nucleotide strandsbetween the first nucleic acid molecule and a defined position of thesecond nucleic acid molecule. Thus, the first nucleic acid willgenerally have a nucleotide sequence that is sufficiently complementaryto a portion of the second nucleic acid molecule to promote nucleotidebase pairing.

[0195] Homologous recombination requires homologous sequences in the tworecombining partner nucleic acids but does not require any specificsequences. As indicated above, site-specific recombination that occurs,for example, at recombination sites such as att sites, is not consideredto be “homologous recombination,” as the phrase is used herein.

[0196] Vector: As used herein, the term “vector” refers to a nucleicacid molecule (preferably DNA) that provides a useful biological orbiochemical property to an insert. A vector may be a nucleic acidmolecule comprising all or a portion of a viral genome. Examples includeplasmids, phages, autonomously replicating sequences (ARS), centromeres,and other sequences that are able to replicate or be replicated in vitroor in a host cell, or to convey a desired nucleic acid segment to adesired location within a host cell. A vector can have one or morerecognition sites (e.g., two, three, four, five, seven, ten, etc.recombination sites, restriction sites, and/or topoisomerases sites) atwhich the sequences can be manipulated in a determinable fashion withoutloss of an essential biological function of the vector, and into which anucleic acid fragment can be spliced in order to bring about itsreplication and cloning. Vectors can further provide primer sites (e.g.,for PCR), transcriptional and/or translational initiation and/orregulation sites, recombinational signals, replicons, selectablemarkers, etc. Clearly, methods of inserting a desired nucleic acidfragment that do not require the use of recombination, transpositions orrestriction enzymes (such as, but not limited to, uracil N-glycosylase(UDG) cloning of PCR fragments (U.S. Pat. Nos. 5,334,575 and 5,888,795,both of which are entirely incorporated herein by reference), T:Acloning, and the like) can also be applied to clone a fragment into acloning vector to be used according to the present invention. Thecloning vector can further contain one or more selectable markers (e.g.,two, three, four, five, seven, ten, etc.) suitable for use in theidentification of cells transformed with the cloning vector.

[0197] Subcloning Vector: As used herein, the phrase “subcloning vector”refers to a cloning vector comprising a circular or linear nucleic acidmolecule that includes, preferably, an appropriate replicon. In thepresent invention, the subcloning vector (segment D in FIG. 1) can alsocontain functional and/or regulatory elements that are desired to beincorporated into the final product to act upon or with the clonednucleic acid insert (segment A in FIG. 1). The subcloning vector canalso contain a selectable marker (preferably DNA).

[0198] Vector Donor: As used herein, the phrase “Vector Donor” refers toone of the two parental nucleic acid molecules (e.g., RNA or DNA) of thepresent invention that carries the nucleic acid segments comprising thenucleic acid vector that is to become part of the desired Product. TheVector Donor comprises a subcloning vector D (or it can be called thecloning vector if the Insert Donor does not already contain a cloningvector) and a segment C flanked by recombination sites (see FIG. 1).Segments C and/or D can contain elements that contribute to selectionfor the desired Product daughter molecule, as described above forselection schemes. The recombination signals can be the same ordifferent, and can be acted upon by the same or different recombinases.In addition, the Vector Donor can be linear or circular. A Vector Donormay be referred to as a Destination Vector.

[0199] Primer: As used herein, the term “primer” refers to a singlestranded or double stranded oligonucleotide that is extended by covalentbonding of nucleotide monomers during amplification or polymerization ofa nucleic acid molecule (e.g., a DNA molecule). In one aspect, theprimer may be a sequencing primer (for example, a universal sequencingprimer). In another aspect, the primer may comprise a recombination siteor portion thereof.

[0200] Adapter: As used herein, the term “adapter” refers to anoligonucleotide or nucleic acid fragment or segment (preferably DNA)that comprises one or more recombination sites (or portions of suchrecombination sites) that can be added to a circular or linear InsertDonor molecule as well as to other nucleic acid molecules describedherein. When using portions of recombination sites, the missing portionmay be provided by the Insert Donor molecule. Such adapters may be addedat any location within a circular or linear molecule, although theadapters are preferably added at or near one or both termini of a linearmolecule. Preferably, adapters are positioned to be located on bothsides (flanking) a particular nucleic acid molecule of interest. Inaccordance with the invention, adapters may be added to nucleic acidmolecules of interest by standard recombinant techniques (e.g.,restriction digest and ligation). For example, adapters may be added toa circular molecule by first digesting the molecule with an appropriaterestriction enzyme, adding the adapter at the cleavage site andreforming the circular molecule that contains the adapter(s) at the siteof cleavage. In other aspects, adapters may be added by homologousrecombination, by integration of RNA molecules, and the like.Alternatively, adapters may be ligated directly to one or more andpreferably both termini of a linear molecule thereby resulting in linearmolecule(s) having adapters at one or both termini. In one aspect of theinvention, adapters may be added to a population of linear molecules,(e.g., a cDNA library or genomic DNA that has been cleaved or digested)to form a population of linear molecules containing adapters at one andpreferably both termini of all or substantial portion of saidpopulation.

[0201] Adapter-Primer: As used herein, the phrase “adapter-primer”refers to a primer molecule that comprises one or more recombinationsites (or portions of such recombination sites) that can be added to acircular or to a linear nucleic acid molecule described herein. Whenusing portions of recombination sites, the missing portion may beprovided by a nucleic acid molecule (e.g., an adapter) of the invention.Such adapter-primers may be added at any location within a circular orlinear molecule, although the adapter-primers are preferably added at ornear one or both termini of a linear molecule. Such adapter-primers maybe used to add one or more recombination sites or portions thereof tocircular or linear nucleic acid molecules in a variety of contexts andby a variety of techniques, including but not limited to amplification(e.g., PCR), ligation (e.g., enzymatic or chemical/synthetic ligation),recombination (e.g., homologous or non-homologous (illegitimate)recombination) and the like.

[0202] Template: As used herein, the term “template” refers to a doublestranded or single stranded nucleic acid molecule that is to beamplified, synthesized or sequenced. In the case of a double-strandedDNA molecule, denaturation of its strands to form a first and a secondstrand is preferably performed before these molecules may be amplified,synthesized or sequenced, or the double stranded molecule may be useddirectly as a template. For single stranded templates, a primercomplementary to at least a portion of the template hybridizes underappropriate conditions and one or more polypeptides having polymeraseactivity (e.g., two, three, four, five, or seven DNA polymerases and/orreverse transcriptases) may then synthesize a molecule complementary toall or a portion of the template. Alternatively, for double strandedtemplates, one or more transcriptional regulatory sequences (e.g., two,three, four, five, seven or more promoters) may be used in combinationwith one or more polymerases to make nucleic acid moleculescomplementary to all or a portion of the template. The newly synthesizedmolecule, according to the invention, may be of equal or shorter lengthcompared to the original template. Mismatch incorporation or strandslippage during the synthesis or extension of the newly synthesizedmolecule may result in one or a number of mismatched base pairs. Thus,the synthesized molecule need not be exactly complementary to thetemplate. Additionally, a population of nucleic acid templates may beused during synthesis or amplification to produce a population ofnucleic acid molecules typically representative of the original templatepopulation.

[0203] Incorporating: As used herein, the term “incorporating” meansbecoming a part of a nucleic acid (e.g., DNA) molecule or primer.

[0204] Library: As used herein, the term “library” refers to acollection of nucleic acid molecules (circular or linear). In oneembodiment, a library may comprise a plurality of nucleic acid molecules(e.g., two, three, four, five, seven, ten, twelve, fifteen, twenty,thirty, fifty, one hundred, two hundred, five hundred one thousand, fivethousand, or more), that may or may not be from a common sourceorganism, organ, tissue, or cell. In another embodiment, a library isrepresentative of all or a portion or a significant portion of thenucleic acid content of an organism (a “genomic” library), or a set ofnucleic acid molecules representative of all or a portion or asignificant portion of the expressed nucleic acid molecules (a cDNAlibrary or segments derived therefrom) in a cell, tissue, organ ororganism. A library may also comprise nucleic acid molecules havingrandom sequences made by de novo synthesis, mutagenesis of one or morenucleic acid molecules, and the like. Such libraries may or may not becontained in one or more vectors (e.g., two, three, four, five, seven,ten, twelve, fifteen, twenty, thirty, fifty, etc.).

[0205] Amplification: As used herein, the term “amplification” refers toany in vitro method for increasing the number of copies of a nucleicacid molecule with the use of one or more polypeptides having polymeraseactivity (e.g., one, two, three, four or more nucleic acid polymerasesor reverse transcriptases). Nucleic acid amplification results in theincorporation of nucleotides into a DNA and/or RNA molecule or primerthereby forming a new nucleic acid molecule complementary to a template.The formed nucleic acid molecule and its template can be used astemplates to synthesize additional nucleic acid molecules. As usedherein, one amplification reaction may consist of many rounds of nucleicacid replication. DNA amplification reactions include, for example,polymerase chain reaction (PCR). One PCR reaction may consist of 5 to100 cycles of denaturation and synthesis of a DNA molecule.

[0206] Nucleotide: As used herein, the term “nucleotide” refers to abase-sugar -phosphate combination. Nucleotides are monomeric units of anucleic acid molecule (DNA and RNA). The term nucleotide includesribonucleoside triphosphates ATP, UTP, CTG, GTP and deoxyribonucleosidetriphosphates such as dATP, dCTP, dITP, dUTP, dGTP, dTTP, or derivativesthereof. Such derivatives include, for example, [α-S]dATP, 7-deaza-dGTPand 7-deaza-dATP. The term nucleotide as used herein also refers todideoxyribonucleoside triphosphates (ddNTPs) and their derivatives.Illustrated examples of dideoxyribonucleoside triphosphates include, butare not limited to, ddATP, ddCTP, ddGTP, ddITP, and ddTTP. According tothe present invention, a “nucleotide” may be unlabeled or detectablylabeled by well known techniques. Detectable labels include, forexample, radioactive isotopes, fluorescent labels, chemiluminescentlabels, bioluminescent labels and enzyme labels.

[0207] Nucleic Acid Molecule: As used herein, the phrase “nucleic acidmolecule” refers to a sequence of contiguous nucleotides (riboNTPs,dNTPs, ddNTPs, or combinations thereof) of any length. A nucleic acidmolecule may encode a full-length polypeptide or a fragment of anylength thereof, or may be non-coding. As used herein, the terms “nucleicacid molecule” and “polynucleotide” may be used interchangeably andinclude both RNA and DNA.

[0208] Oligonucleotide: As used herein, the term “oligonucleotide”refers to a synthetic or natural molecule comprising a covalently linkedsequence of nucleotides that are joined by a phosphodiester bond betweenthe 3′ position of the pentose of one nucleotide and the 5′ position ofthe pentose of the adjacent nucleotide.

[0209] Polypeptide: As used herein, the term “polypeptide” refers to asequence of contiguous amino acids of any length. The terms “peptide,”“oligopeptide,” or “protein” may be used interchangeably herein with theterm “polypeptide.”

[0210] Hybridization: As used herein, the terms “hybridization” and“hybridizing” refer to base pairing of two complementary single-strandednucleic acid molecules (RNA and/or DNA) to give a double strandedmolecule. As used herein, two nucleic acid molecules may hybridize,although the base pairing is not completely complementary. Accordingly,mismatched bases do not prevent hybridization of two nucleic acidmolecules provided that appropriate conditions, well known in the art,are used. In some aspects, hybridization is said to be under “stringentconditions.” By “stringent conditions,” as the phrase is used herein, ismeant overnight incubation at 42° C. in a solution comprising: 50%formamide, 5×SSC (750 mM NaCl, 75 mM trisodium citrate), 50 mM sodiumphosphate (pH 7.6), 5×Denhardt's solution, 10% dextran sulfate, and 20μg/ml denatured, sheared salmon sperm DNA, followed by washing thefilters in 0.1×SSC at about 65° C.

[0211] Transduce: As used herein, “transduce” and “transduction” referto a process of introducing a virus into a cell type that does notsupport replication of the virus and does not result in the productionof infectious viral progeny. In contrast, “infect” or “infection” areused to indicate introduction of a virus into a cell type that supportsreplication and results in the production of infectious viral progeny.

[0212] Other terms used in the fields of recombinant nucleic acidtechnology and molecular and cell biology as used herein will begenerally understood by one of ordinary skill in the applicable arts.

[0213] Overview

[0214] The present invention relates to methods, compositions and kitsfor the recombinational joining of two or more segments or nucleic acidmolecules to produce a nucleic acid molecule comprising all or a portionof a viral genome, for example, a recombinant viral vector. Further, thepresent invention relates to methods, compositions and kits for thetopoisomerase-mediated joining of two or more segments or nucleic acidmolecules to produce a nucleic acid molecule comprising all or a portionof a viral genome, for example, a recombinant viral vector. The presentinvention also relates to methods, compositions and kits for the joiningby other means (e.g., ligase) of two or more segments or nucleic acidmolecules to produce a nucleic acid molecule comprising all or a portionof a viral genome, for example, a recombinant viral vector. Theinvention also includes methods for preparing such nucleic acidmolecules, as well as compositions comprising such nucleic acidmolecules.

[0215] The present invention also contemplates methods for using thesemolecules to generate host cells, methods of using these molecules toproduce polypeptide and/or RNA expression products.

[0216] In one embodiment, at least two nucleic acid segments, eachcomprising at least one recombination site, are contacted with suitablerecombination proteins to effect the joining of all or a portion of thetwo molecules, depending on the position in the molecules of therecombination sites that undergo recombination. Each individual nucleicacid segment may comprise a variety of sequences including, but notlimited to viral sequences, sequences suitable for use as primer bindingsites (e.g., sequences for which a primer such as a sequencing primer oramplification primer may hybridize to initiate nucleic acid synthesis,amplification or sequencing), transcription or translation signals orregulatory sequences such as promoters and/or enhancers, ribosomalbinding sites, Kozak sequences, start codons, termination signals suchas stop codons, origins of replication, recombination sites (or portionsthereof), selectable markers, and genes or portions of genes to createprotein fusions (e.g., N-terminal or C-terminal) such as GST, GUS, GFP,YFP, CFP, maltose binding protein, 6 histidines (HIS6), epitopes,haptens and the like and combinations thereof. The vectors used forcloning such segments may also comprise these functional sequences(e.g., promoters, primer sites, etc.).

[0217] After joining of the segments, the product molecule will oftencontain at least sufficient viral sequences to permit the packaging ofthe product molecule in a viral particle. In the case where the viralsequences are adenoviral sequences, the product molecule may contain aleft ITR, a packaging sequence and a right ITR, and/or sufficient othersequences to result in a molecule of appropriate size for packaging. Insome embodiments, the product molecule comprises sufficient viralsequences to be an infectious viral genome when introduced into apermissive host cell. In some embodiments, a recombinant adenoviralvector produced by the methods of the invention may comprise a left ITR,a packaging sequence a first recombination site, a sequence of interest,a second recombination site, and additional adenoviral sequencesincluding a right ITR. In the case where the viral sequences areretroviral sequences, the product molecule may contain a 5′-LTR, a3′-LTR and a packaging sequence (Ψ), and/or sufficient other sequencesto result in a molecule of appropriate size for packaging. In someembodiments, the product molecule comprises sufficient retroviralsequences to integrate into the genome of host cell into which it isintroduced but not enough viral sequences to produce an infectious virusin the host cell. In some embodiments, a recombinant retroviral vectorproduced by the methods of the invention may be a plasmid comprising a5′-LTR, a packaging sequence a first recombination site, a sequence ofinterest, and a second recombination site, and additional retroviralsequences including a 3′-LTR.

[0218] Recombination Sites

[0219] Recombination sites for use in the invention may be any nucleicacid that can serve as a substrate in a recombination reaction. Suchrecombination sites may be wild-type or naturally occurringrecombination sites, or modified, variant, derivative, or mutantrecombination sites. Examples of recombination sites for use in theinvention include, but are not limited to, phage-lambda recombinationsites (such as attP, attB, attL, and attR and mutants or derivativesthereof) and recombination sites from other bacteriophages such asphi80, P22, P2, 186, P4 and P1 (including lox sites such as loxP andloxP511).

[0220] In some embodiments, recombination sites that may be used in thepractice of the invention include recombination sites that undergorecombination with compatible recombination sites in the presence of oneor more recombination proteins active in the phage lambda recombinationsystem, for example, one or more of Int, IHF, FIS, and/or Xis. Theinvention also contemplates nucleic acid molecules comprising suchrecombination sites and compositions comprising such nucleic acidmolecules. Preferred recombination proteins and mutant, modified,variant, or derivative recombination sites for use in the inventioninclude those described in U.S. Pat. Nos. 5,888,732, 6,143,557,6,171,861, 6,270,969, and 6,277,608 and in U.S. application Ser. No.09/438,358 (filed Nov. 12, 1999), based upon U.S. provisionalapplication No. 60/108,324 (filed Nov. 13, 1998). Mutated att sites(e.g., attB 1-10, attP 1-10, attR 1-10 and attL 1-10) are described inU.S. application Ser. No. 09/517,466, filed Mar. 2, 2000, and Ser. No.09/732,914, filed Dec. 11, 2000 (published as 2002 0007051-A1) thedisclosures of which are specifically incorporated herein by referencein their entirety. Other suitable recombination sites and proteins arethose associated with the GATEWAY™ Cloning Technology available fromInvitrogen Corporation, Carlsbad, Calif., and described in the productliterature of the GATEWAY™ Cloning Technology, the entire disclosures ofall of which are specifically incorporated herein by reference in theirentireties.

[0221] Sites that may be used in the present invention include attsites. The 15 bp core region of the wildtype att site (GCTTTTTTAT ACTAA(SEQ ID NO:)), which is identical in all wildtype att sites, may bemutated in one or more positions. Other att sites that specificallyrecombine with other att sites can be constructed by alteringnucleotides in and near the 7 base pair overlap region, bases 6-12 ofthe core region. Thus, recombination sites suitable for use in themethods, molecules, compositions, and vectors of the invention include,but are not limited to, those with insertions, deletions orsubstitutions of one, two, three, four, or more nucleotide bases withinthe 15 base pair core region (see U.S. application Ser. No. 08/663,002,filed Jun. 7, 1996 (now U.S. Pat. No. 5,888,732) and Ser. No.09/177,387, filed Oct. 23, 1998, which describes the core region infurther detail, and the disclosures of which are incorporated herein byreference in their entireties). Recombination sites suitable for use inthe methods, compositions, and vectors of the invention also includethose with insertions, deletions or substitutions of one, two, three,four, or more nucleotide bases within the 15 base pair core region thatare at least 50% identical, at least 55% identical, at least 60%identical, at least 65% identical, at least 70% identical, at least 75%identical, at least 80% identical, at least 85% identical, at least 90%identical, or at least 95% identical to this 15 base pair core region.

[0222] As a practical matter, whether any particular nucleic acidmolecule is at least 50%, 60%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%,98% or 99% identical to, for instance, a given recombination sitenucleotide sequence or portion thereof can be determined conventionallyusing known computer programs such as DNAsis software (Hitachi Software,San Bruno, Calif.) for initial sequence alignment followed by ESEEversion 3.0 DNA/protein sequence software (cabot@trog.mbb.sfu.ca) formultiple sequence alignments. Alternatively, such determinations may beaccomplished using the BESTFIT program (Wisconsin Sequence AnalysisPackage, Genetics Computer Group, University Research Park, 575 ScienceDrive, Madison, Wis. 53711), which employs a local homology algorithm(Smith and Waterman, Advances in Applied Mathematics 2: 482-489 (1981))to find the best segment of homology between two sequences. When usingDNAsis, ESEE, BESTFIT or any other sequence alignment program todetermine whether a particular sequence is, for instance, 95% identicalto a reference sequence according to the present invention, theparameters are set such that the percentage of identity is calculatedover the full length of the reference nucleotide sequence and that gapsin homology of up to 5% of the total number of nucleotides in thereference sequence are allowed. Computer programs such as thosediscussed above may also be used to determine percent identity andhomology between two proteins at the amino acid level.

[0223] Analogously, the core regions in attB1, attP1, attL1 and attR1are identical to one another, as are the core regions in attB2, attP2,attL2 and attR2. Nucleic acid molecules suitable for use with theinvention also include those comprising insertions, deletions orsubstitutions of one, two, three, four, or more nucleotides within theseven base pair overlap region (TTTATAC, bases 6-12 in the core region).The overlap region is defined by the cut sites for the integrase proteinand is the region where strand exchange takes place. Examples of suchmutants, fragments, variants and derivatives include, but are notlimited to, nucleic acid molecules in which (1) the thymine at position1 of the seven bp overlap region has been deleted or substituted with aguanine, cytosine, or adenine; (2) the thymine at position 2 of theseven bp overlap region has been deleted or substituted with a guanine,cytosine, or adenine; (3) the thymine at position 3 of the seven bpoverlap region has been deleted or substituted with a guanine, cytosine,or adenine; (4) the adenine at position 4 of the seven bp overlap regionhas been deleted or substituted with a guanine, cytosine, or thymine;(5) the thymine at position 5 of the seven bp overlap region has beendeleted or substituted with a guanine, cytosine, or adenine; (6) theadenine at position 6 of the seven bp overlap region has been deleted orsubstituted with a guanine, cytosine, or thymine; and (7) the cytosineat position 7 of the seven bp overlap region has been deleted orsubstituted with a guanine, thymine, or adenine; or any combination ofone or more (e.g., two, three, four, five, etc.) such deletions and/orsubstitutions within this seven bp overlap region. The nucleotidesequences of representative seven base pair core regions are set outbelow.

[0224] Altered att sites have been constructed that demonstrate that (1)substitutions made within the first three positions of the seven basepair overlap (TTTATAC) strongly affect the specificity of recombination,(2) substitutions made in the last four positions (TTTATAC) onlypartially alter recombination specificity, and (3) nucleotidesubstitutions outside of the seven bp overlap, but elsewhere within the15 base pair core region, do not affect specificity of recombination butdo influence the efficiency of recombination. Thus, nucleic acidmolecules and methods of the invention include those comprising oremploying one, two, three, four, five, six, eight, ten, or morerecombination sites which affect recombination specificity, particularlyone or more (e.g., one, two, three, four, five, six, eight, ten, twenty,thirty, forty, fifty, etc.) different recombination sites that maycorrespond substantially to the seven base pair overlap within the 15base pair core region, having one or more mutations that affectrecombination specificity. Particularly preferred such molecules maycomprise a consensus sequence such as NNNATAC wherein “N” refers to anynucleotide (i.e., may be A, G, T/U or C). Preferably, if one of thefirst three nucleotides in the consensus sequence is a T/U, then atleast one of the other two of the first three nucleotides is not a T/U.

[0225] The core sequence of each att site (attB, attP, attL and attR)can be divided into functional units consisting of integrase bindingsites, integrase cleavage sites and sequences that determinespecificity. Specificity determinants are defined by the first threepositions following the integrase top strand cleavage site. These threepositions are shown with underlining in the following referencesequence: CAACTTTTTTATAC AAAGTTG (SEQ ID NO:______). Modification ofthese three positions (64 possible combinations) can be used to generateatt sites that recombine with high specificity with other att siteshaving the same sequence for the first three nucleotides of the sevenbase pair overlap region. The possible combinations of first threenucleotides of the overlap region are shown in Table 1. TABLE 1Modifications of the First Three Nucleotides of the att Site Seven BasePair Overlap Region that Alter Recombination Specificity. AAA CAA GAATAA AAC CAC GAC TAC AAG CAG GAG TAG AAT CAT GAT TAT ACA CCA GCA TCA ACCCCC GCC TCC ACG CCG GCG TCG ACT CCT GCT TCT AGA CGA GGA TGA AGC CGC GGCTGC AGG CGG GGG TGG AGT CGT GGT TGT ATA CTA GTA TTA ATC CTC GTC TTC ATGCTG GTG TTG ATT CTT GTT TTT

[0226] Representative examples of seven base pair att site overlapregions suitable for in methods, compositions and vectors of theinvention are shown in Table 2. The invention further includes nucleicacid molecules comprising one or more (e.g., one, two, three, four,five, six, eight, ten, twenty, thirty, forty, fifty, etc.) nucleotidessequences set out in Table 2. Thus, for example, in one aspect, theinvention provides nucleic acid molecules comprising the nucleotidesequence GAAATAC, GATATAC, ACAATAC, or TGCATAC. TABLE 2 RepresentativeExamples of Seven Base Pair att Site Overlap Regions Suitable for use inthe recombination sites of the Invention. AAAATAC CAAATAC GAAATACTAAATAC AACATAC CACATAC GACATAC TACATAC AAGATAC CAGATAC GAGATAC TAGATACAATATAC CATATAC GATATAC TATATAC ACAATAC CCAATAC GCAATAC TCAATAC ACCATACCCCATAC GCCATAC TCCATAC ACGATAC CCGATAC GCGATAC TCGATAC ACTATAC CCTATACGCTATAC TCTATAC AGAATAC CGAATAC GGAATAC TGAATAC AGCATAC CGCATAC GGCATACTGCATAC AGGATAC CGGATAC GGGATAC TGGATAC AGTATAC CGTATAC GGTATAC TGTATACATAATAC CTAATAC GTAATAC TTAATAC ATCATAC CTCATAC GTCATAC TTCATAC ATGATACCTGATAC GTGATAC TTGATAC ATTATAC CTTATAC GTTATAC TTTATAC

[0227] As noted above, alterations of nucleotides located 3′ to thethree base pair region discussed above can also affect recombinationspecificity. For example, alterations within the last four positions ofthe seven base pair overlap can also affect recombination specificity.

[0228] For example, mutated att sites that may be used in the practiceof the present invention include attB1 (AGCCTGCTTT TTTGTACAAA CTTGT (SEQID NO: )), attP1 (TACAGGTCAC TAATACCATC TAAGTAGTTG ATTCATAGTG ACTGGATATGTTGTGTTTTA CAGTATTATG TAGTCTGTTT TTTATGCAAA ATCTAATTTA ATATATTGATATTTATATCA TTTTACGTTT CTCGTTCAGC TTTTTTGTAC AAAGTTGGCA TTATAAAAAAGCATTGCTCA TCAATTTGTT GCAACGAACA GGTCACTATC AGTCAAAATA AAATCATTAT TTG(SEQ ID NO: )), attL1 (CAAATAATGA TTTTATTTTG ACTGATAGTG ACCTGTTCGTTGCAACAAAT TGATAAGCAA TGCTTTTTTA TAATGCCAAC TTTGTACAAA AAAGCAGGCT (SEQID NO:)), and attR1 (ACAAGTTTGT ACAAAAAAGC TGAACGAGAA ACGTAAAATGATATAAATAT CAATATATTA AATTAGATTT TGCATAAAAA ACAGACTACA TAATACTGTAAAACACAACA TATCCAGTCA CTATG (SEQ ID NO: )). Table 3 provides thesequences of the regions surrounding the core region for the wild typeatt sites (attB0, P0, R0, and L0) as well as a variety of other suitablerecombination sites. Those skilled in the art will appreciated that theremainder of the site may be the same as the corresponding site (B, P,L, or R) listed above. TABLE 3 Nucleotide sequences of att sites. attB0AGCCTGCTTT TTTATACTAA CTTGAGC (SEQ ID NO: ) attP0 GTTCAGCTTT TTTATACTAAGTTGGCA (SEQ ID NO: ) attL0 AGCCTGCTTT TTTATACTAA GTTGGCA (SEQ ID NO: )attR0 GTTCAGCTTT TTTATACTAA CTTGAGC (SEQ ID NO: ) attB1 AGCCTGCTTTTTTGTACAAA CTTGT (SEQ ID NO: ) attP1 GTTCAGCTTT TTTGTACAAA GTTGGCA (SEQID NO: ) attL1 AGCCTGCTTT TTTGTACAAA GTTGGCA (SEQ ID NO: ) attR1GTTCAGCTTT TTTGTACAAA CTTGT (SEQ ID NO: ) attB2 ACCCAGCTTT CTTGTACAAAGTGGT (SEQ ID NO: ) attP2 GTTCAGCTTT CTTGTACAAA GTTGGCA (SEQ ID NO: )attL2 ACCCAGCTTT CTTGTACAAA GTTGGCA (SEQ ID NO: ) attR2 GTTCAGCTTTCTTGTACAAA GTGGT (SEQ ID NO: ) attB5 CAACTTTATT ATACAAAGTT GT (SEQ IDNO: ) attP5 GTTCAACTTT ATTATACAAA GTTGGCA (SEQ ID NO: ) attL5 CAACTTTATTATACAAAGTT GGCA (SEQ ID NO: ) attR5 GTTCAACTTT ATTATACAAA GTTGT (SEQ IDNO: ) attB11 CAACTTTTCT ATACAAAGTT GT (SEQ ID NO: ) attP11 GTTCAACTTTTCTATACAAA GTTGGCA (SEQ ID NO: ) attL11 CAACTTTTCT ATACAAAGTT GGCA (SEQID NO: ) attR11 GTTCAACTTT TCTATACAAA GTTGT (SEQ ID NO: ) attB17CAACTTTTGT ATACAAAGTT GT (SEQ ID NO: ) attP17 GTTCAACTTT TGTATACAAAGTTGGCA (SEQ ID NO: ) attL17 CAACTTTTGT ATACAAAGTT GGCA (SEQ ID NO: )attR17 GTTCAACTTT TGTATACAAA GTTGT (SEQ ID NO: ) attB19 CAACTTTTTCGTACAAAGTT GT (SEQ ID NO: ) attP19 GTTCAACTTT TTCGTACAAA GTTGGCA (SEQ IDNO: ) attL19 CAACTTTTTC GTACAAAGTT GGCA (SEQ ID NO: ) attR19 GTTCAACTTTTTCGTACAAA GTTGT (SEQ ID NO: ) attB20 CAACTTTTTG GTACAAAGTT GT (SEQ IDNO: ) attP20 GTTCAACTTT TTGGTACAAA GTTGGCA (SEQ ID NO: ) attL20CAACTTTTTG GTACAAAGTT GGCA (SEQ ID NO: ) attR20 GTTCAACTTT TTGGTACAAAGTTGT (SEQ ID NO: ) attB21 CAACTTTTTA ATACAAAGTT GT (SEQ ID NO: ) attP21GTTCAACTTT TTAATACAAA GTTGGCA (SEQ ID NO: ) attL21 CAACTTTTTA ATACAAAGTTGGCA (SEQ ID NO: ) attR21 GTTCAACTTT TTAATACAAA GTTGT (SEQ ID NO: )

[0229] Other recombination sites having unique specificity (i.e., afirst site will recombine with its corresponding site and will notsubstantially recombine with a second site having a differentspecificity) are known to those skilled in the art and may be used topractice the present invention.

[0230] Corresponding recombination proteins for these systems may beused in accordance with the invention with the indicated recombinationsites. Other systems providing recombination sites and recombinationproteins for use in the invention include the FLP/FRT system fromSaccharomyces cerevisiae, the resolvase family (e.g., γδ, TndX, TnpX,Tn3 resolvase, Hin, Hjc, Gin, SpCCE1, ParA, and Cin), and IS231 andother Bacillus thuringiensis transposable elements. Other suitablerecombination systems for use in the present invention include the XerCand XerD recombinases and the psi, dif and cer recombination sites in E.coli. Other suitable recombination sites may be found in U.S. Pat. No.5,851,808 issued to Elledge and Liu which is specifically incorporatedherein by reference.

[0231] The materials and methods of the invention may further encompassthe use of “single use” recombination sites which undergo recombinationone time and then either undergo recombination with low frequency (e.g.,have at least five fold, at least ten fold, at least fifty fold, atleast one hundred fold, or at least one thousand fold lowerrecombination activity in subsequent recombination reactions) or areessentially incapable of undergoing recombination. The invention alsoprovides methods for making and using nucleic acid molecules whichcontain such single use recombination sites and molecules which containthese sites. Examples of methods which can be used to generate andidentify such single use recombination sites are set out below. Furtherexamples of methods which can be used to generate and identify suchsingle use recombination sites are set out in PCT/US00/21623, publishedas WO 01/11058, which claims priority to U.S. provisional patentapplication 60/147,892, filed Aug. 9, 1999, both of which arespecifically incorporated herein by reference.

[0232] The att system core integrase binding site comprises aninterrupted seven base pair inverted repeat having the followingnucleotide sequence:

[0233] ------> . . . <------

[0234] caactttnnnnnnnaaagttg (SEQ ID NO:39),

[0235] as well as variations thereof which can comprise either perfector imperfect repeats.

[0236] The repeat elements can be subdivided into two distal and/orproximal “domains” composed of caac/gttg segments (underlined), whichare distal to the central undefined sequence (the nucleotides of whichare represented by the letter “n”), and ttt/aaa segments, which areproximal to the central undefined sequence.

[0237] Alterations in the sequence composition of the distal and/orproximal domains on one or both sides of the central undefined regioncan affect the outcome of a recombination reaction. The scope and scaleof the effect is a function of the specific alterations made, as well asthe particular recombinational event (e.g., LR vs. BP reactions).

[0238] For example, it is believed that an attB site altered to have thefollowing nucleotide sequence:

[0239] ------> . . . <------

[0240] caactttnnnnnnnaaacaag (SEQ ID NO:40),

[0241] will functionally interact with a cognate attP and generate attLand attR. However, whichever of the latter two recombination sitesacquires the segment containing “caag” (located on the left side of thesequence shown above) will be rendered non-functional to subsequentrecombination events. The above is only one of many possible alterationsin the core integrase binding sequence which can render att sitesnon-functional after engaging in a single recombination event. Thus,single use recombination sites may be prepared by altering nucleotidesin the seven base pair inverted repeat regions which abut seven basepair overlap regions of att sites. This region is representedschematically as:

CAAC TTT [Seven Base Pair Overlap Region] AAA GTTG.

[0242] In generating single use recombination sites, one, two, three,four or more of nucleotides of the sequences CAACTTT or AAAGTTG (i.e.,the seven base pair inverted repeat regions) may be substituted withother nucleotides or deleted altogether. These seven base pair invertedrepeat regions represent complementary sequences with respect to eachother. Thus, alterations may be made in either seven base pair invertedrepeat region in order to generate single use recombination sites.Further, when DNA is double stranded and one seven base pair invertedrepeat region is present, the other seven base pair inverted repeatregion will also be present on the other strand.

[0243] Using the sequence CAACTTT for illustration, examples of sevenbase pair inverted repeat regions which can form single userecombination sites include, but are not limited to, nucleic acidmolecules in which (1) the cytosine at position 1 of the seven base pairinverted repeat region has been deleted or substituted with a guanine,adenine, or thymine; (2) the adenine at position 2 of the seven basepair inverted repeat region has been deleted or substituted with aguanine, cytosine, or thymine; (3) the adenine at position 3 of theseven base pair inverted repeat region has been deleted or substitutedwith a guanine, cytosine, or thymine; (4) the cytosine at position 4 ofthe seven base pair inverted repeat region has been deleted orsubstituted with a guanine, adenine, or thymine; (5) the thymine atposition 5 of the seven base pair inverted repeat region has beendeleted or substituted with a guanine, cytosine, or adenine; (6) thethymine at position 6 of the seven base pair inverted repeat region hasbeen deleted or substituted with a guanine, cytosine, or adenine; and(7) the thymine at position 7 of the seven base pair inverted repeatregion has been deleted or substituted with a guanine, cytosine, oradenine; or any combination of one, two, three, four, or more suchdeletions and/or substitutions within this seven base pair region.Representative examples of nucleotide sequences of the above describedseven base pair inverted repeat regions are set out below in Table 4.TABLE 4 Representative examples of nucleotide sequences of seven basepair inverted repeat regions. aagaaaa aagagcg aagagaa aagatat ccgccacccgcctc ccgcaca ccgcttt ggtggga ggtgctc ggtgata ggtgtat ttctttg ttctctcttctgaa ttctttt aatacac aatagcg aataaca aatatat cctcgga cctcccg cctcacacctcttt ggcgaaa ggcgccg ggcggaa ggcgtat ttgtcac ttgtgcg ttgtaca ttgttttacaagga acaaccg acaaata acaattt caccttg caccaga caccgaa cacctat gaggcacgagggcg gaggaca gaggttt tattgga tattaga tattaca tatttat agaaaaa agaaagaagaagaa agaattt cgcccac cgccctc cgccaca cgccttt gcgggga gcgggcg gcggatagcggtat tcttttg tcttccg tcttgaa tcttttt ataacac ataactc ataaaca ataatttctccaaa ctccgcg ctccata ctcctat gtgggga gtggccg gtgggaa gtggtat tgttttgtgttctc tgttaca tgttttt

[0244] Representative examples of nucleotide sequences which form singleuse recombination sites may also be prepared by combining a nucleotidesequence set out in Table 5, Section 1, with a nucleotide sequence setout in Table 5, Section 2. Single use recombination sites may also beprepared by the insertion of one or more (e.g., one, two, three, four,five six, seven, etc.) nucleotides internally within these regions.TABLE 5 Representative examples of nucleotide sequences which formsingle use recombination sites. Section 1 (CAAG) Section 2 (TTT) aaaacccc gggg tttt aaa cca ttc aaac ccca ggga ttta aac cac ttg aaag ccctgggc tttc aag cgc tat aaat cccg gggt tttg aat ctc tct aaca ccac ggagttat aca ggg tgt aaga ccgc ggtg ttct aga gga aata cctc ggcg ttgt ata ggcacaa cacc gagg tatt caa ggt agaa cgcc gcgg tctt gaa gag ataa ctcc gtggtgtt taa gcg caaa accc aggg attt ccc gtg gaaa gccc CGG  cttt ccg ttttaaa tccc tggg gttt cct tta

[0245] In most instances where one seeks to prevent recombination eventswith respect to a particular nucleic acid segment, the altered sequencewill be located proximally to the nucleic acid segment. Using thefollowing schematic for illustration:

=5′ Nucleic Acid Segment 3′=caac ttt (Seven Base Pair Overlap Region)AAA GTTG,

[0246] the lower case nucleotide sequence which represent a seven basepair inverted repeat region (i.e., caac ttt) will generally have asequence altered by insertion, deletion, and/or substitution to form asingle use recombination site when one seeks to prevent recombination atthe 3′ end (i.e., proximal end with respect to the nucleic acid segment)of the nucleic acid segment shown. Thus, a single recombination reactioncan be used, for example, to integrate the nucleic acid segments intoanother nucleic acid molecule, then the recombination site becomeseffectively non-functional, preventing the site from engaging in furtherrecombination reactions. Similarly, single use recombination sites canbe position at both ends of a nucleic acid segment so that the nucleicacid segment can be integrated into another nucleic acid molecule, orcircularized, and will remain integrated, or circularized even in thepresence of recombinases.

[0247] A number of methods may be used to screen potential single userecombination sites for functional activity (e.g., undergo onerecombination event followed by the failure to undergo subsequentrecombination events). For example, with respect to the screening ofrecombination sites to identify those which become non-functional aftera single recombination event, a first recombination reaction may beperformed to generate a plasmid in which a negative selection marker islinked to one or more potentially defective recombination sites. Theplasmid may then be reacted with another nucleic acid molecule whichcomprises a positive selection marker similarly linked to recombinationsites. Thus, this selection system is designed such that molecules whichrecombine are susceptible to negative selection and molecules which donot recombine may be selected for by positive selection. Using such asystem, one may then directly select for desired single use core sitemutants.

[0248] As one skilled in the art would recognize, any number ofscreening assays may be designed which achieve the same results as thosedescribed above. In many instances, these assays will be designed sothat an initial recombination event takes place and then recombinationsites which are unable to engage in subsequent recombination events areidentified or molecules which contain such recombination sites areselected for. A related screening assay would result in selectionagainst nucleic acid molecule which have undergone a secondrecombination event. Further, as noted above, screening assays can bedesigned where there is selection against molecules which have engagedin subsequent recombination events and selection for those which havenot engaged in subsequent recombination events.

[0249] Single use recombination sites are especially useful for eitherdecreasing the frequency of or preventing recombination when eitherlarge number of nucleic acid segments are attached to each other ormultiple recombination reactions are performed. Thus, the inventionfurther includes nucleic acid molecules which contain single userecombination sites, as well as methods for performing recombinationusing these sites.

[0250] Recombination sites used with the invention may also haveembedded functions or properties. An embedded functionality is afunction or property conferred by a nucleotide sequence in arecombination site that is not directly associated with recombinationefficiency or specificity. For example, recombination sites may containprotein coding sequences (e.g., intein coding sequences), intron/exonsplice sites, origins of replication, and/or stop codons. Further,recombination sites that have more than one (e.g., two, three, four,five, etc.) embedded functions or properties may also be prepared.

[0251] In some instances it will be advantageous to remove either RNAcorresponding to recombination sites from RNA transcripts or amino acidresidues encoded by recombination sites from polypeptides translatedfrom such RNAs. Removal of such sequences can be performed in severalways and can occur at either the RNA or protein level. One instancewhere it may be advantageous to remove RNA transcribed from arecombination site will be when constructing a fusion polypeptidebetween a polypeptide of interest and a coding sequence present on thevector. The presence of an intervening recombination site between theORF of the polypeptide of interest and the vector coding sequences mayresult in the recombination site (1) contributing codons to the mRNAthat result in the inclusion of additional amino acid residues in theexpression product, (2) contributing a stop codon to the mRNA thatprevents the production of the desired fusion protein, and/or (3)shifting the reading frame of the mRNA such that the two protein are notfused “in-frame.”

[0252] In one aspect, the invention provides methods for removingnucleotide sequences encoded by recombination sites from RNA molecules.One example of such a method employs the use of intron/exon splice sitesto remove RNA encoded by recombination sites from RNA transcripts.Nucleotide sequences that encode intron/exon splice sites may be fullyor partially embedded in the recombination sites used in the presentinvention and/or may encoded by adjacent nucleic acid sequence.Sequences to be excised from RNA molecules may be flanked by splicesites that are appropriately located in the sequence of interest and/oron the vector. For example, one intron/exon splice site may be encodedby a recombination site and another intron/exon splice site may beencoded by other nucleotide sequences (e.g., nucleic acid sequences ofthe vector or a nucleic acid of interest). Nucleic acid splicing is wellknown to those skilled in the art and is discussed in the followingpublications: R. Reed, Curr. Opin. Genet. Devel. 6:215-220 (1996); S.Mount, Nucl. Acids. Res. 10:459-472, (1982); P. Sharp, Cell 77:805-815,(1994); K. Nelson and M. Green, Genes and Devel. 23:319-329 (1988); andT. Cooper and W. Mattox, Am. J. Hum. Genet. 61:259-266 (1997).

[0253] Splice sites can be suitably positioned in a number of locations.For example, a Destination Vector designed to express an inserted ORFwith an N-terminal fusion—for example, with a detectable marker—thefirst splice site could be encoded by vector sequences located 3′ to thedetectable marker coding sequences and the second splice site could bepartially embedded in the recombination site that separates thedetectable marker coding sequences from the coding sequences of the ORF.Further, the second splice site either could abut the 3′ end of therecombination site or could be positioned a short distance (e.g., 2, 4,8, 10, 20 nucleotides) 3′ to the recombination site. In addition,depending on the length of the recombination site, the second splicesite could be fully embedded in the recombination site.

[0254] A modification of the method described above involves theconnection of multiple nucleic acid segments that, upon expression,results in the production of a fusion protein. In one specific example,one nucleic acid segment encodes detectable marker—for example, GFP—andanother nucleic acid segment that encodes an ORF of interest. Each ofthese segments is flanked by recombination sites. In addition, thenucleic acid segments that encodes the detectable marker contains anintron/exon splice site near its 3′ terminus and the nucleic acidsegments that contains the ORF of interest also contains an intron/exonsplice site near its 5′ terminus. Upon recombination, the nucleic acidsegment that encodes the detectable marker is positioned 5′ to thenucleic acid segment that encodes the ORF of interest. Further, thesetwo nucleic acid segments are separated by a recombination site that isflanked by intron/exon splice sites. Excision of the interveningrecombination site thus occurs after transcription of the fuision mRNA.Thus, in one aspect, the invention is directed to methods for removingRNA transcribed from recombination sites from transcripts generated fromnucleic acids described herein.

[0255] Splice sites may introduced into nucleic acid molecules to beused in the present invention in a variety of ways. One method thatcould be used to introduce intron/exon splice sites into nucleic acidsegments is PCR. For example, primers could be used to generate nucleicacid segments corresponding to an ORF of interest and containing both arecombination site and an intron/exon splice site.

[0256] The above methods can also be used to remove RNA corresponding torecombination sites when the nucleic acid segment that is recombinedwith another nucleic acid segment encodes RNA that is not produced in atranslatable format. One example of such an instance is where a nucleicacid segment is inserted into a vector in a manner that results in theproduction of antisense RNA. As discussed below, this antisense RNA maybe fused, for example, with RNA that encodes a ribozyme. Thus, theinvention also provides methods for removing RNA corresponding torecombination sites from such molecules.

[0257] The invention further provides methods for removing amino acidsequences encoded by recombination sites from protein expressionproducts by protein splicing. Nucleotide sequences that encode proteinsplice sites may be fully or partially embedded in the recombinationsites that encode amino acid sequences excised from proteins or proteinsplice sites may be encoded by adjacent nucleotide sequences. Similarly,one protein splice site may be encoded by a recombination site andanother protein splice sites may be encoded by other nucleotidesequences (e.g., nucleic acid sequences of the vector or a nucleic acidof interest).

[0258] It has been shown that protein splicing can occur by excision ofan intein from a protein molecule and ligation of flanking segments(see, e.g., Derbyshire, et al., Proc. Natl. Acad. Sci. (USA)95:1356-1357 (1998)). In brief, inteins are amino acid segments that arepost-translationally excised from proteins by a self-catalytic splicingprocess. A considerable number of intein consensus sequences have beenidentified (see, e.g., Perler, Nucleic Acids Res. 27:346-347 (1999)).

[0259] Similar to intron/exon splicing, N- and C-terminal intein motifshave been shown to be involved in protein splicing. Thus, the inventionfurther provides compositions and methods for removing amino acidresidues encoded by recombination sites from protein expression productsby protein splicing. In particular, this aspect of the invention isrelated to the positioning of nucleic acid sequences that encode inteinsplice sites on both the 5′ and 3′ end of recombination sites positionedbetween two coding regions. Thus, when the protein expression product isincubated under suitable conditions, amino acid residues encoded theserecombination sites will be excised.

[0260] Protein splicing may be used to remove all or part of the aminoacid sequences encoded by recombination sites. Nucleic acid sequencethat encode inteins may be fully or partially embedded in recombinationsites or may adjacent to such sites. In certain circumstances, it may bedesirable to remove considerable numbers of amino acid residues beyondthe N- and/or C-terminal ends of amino acid sequences encoded byrecombination sites. In such instances, intein coding sequence may belocated a distance (e.g., 30, 50, 75, 100, etc. nucleotides) 5′ and/or3′ to the recombination site.

[0261] While conditions suitable for intein excision will vary with theparticular intein, as well as the protein that contains this intein,Chong, et al., Gene 192:271-281 (1997), have demonstrated that amodified Saccharomyces cerevisiae intein, referred to as Sce VMA intein,can be induced to undergo self-cleavage by a number of agents including1,4-dithiothreitol (DTT), β-mercaptoethanol, and cysteine. For example,intein excision/splicing can be induced by incubation in the presence of30 mM DTT, at 4° C. for 16 hours.

[0262] Topoisomerase Cloning

[0263] The present invention also relates to methods of using one ormore topoisomerases to generate a recombinant nucleic acid molecules ofthe invention (e.g., molecules comprising all or a portion of a viralgenome such as a viral vector) comprising two or more nucleotidesequences, any one or more of which may comprise, for example, all or aportion of a viral genome. Topoisomerases may be used in combinationwith recombinational cloning techniques described above. For example, atopoisomerase-mediated reaction may be used to attach one or morerecombination sites to one or more nucleic acid segments. The segmentsmay then be further manipulated and combined using, for example,recombinational cloning techniques.

[0264] In one aspect, the present invention provides methods for linkinga first and at least a second nucleic acid segment (either or both ofwhich may contain viral sequences and/or sequences of interest) with atleast one (e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, etc.) topoisomerase(e.g., a type IA, type IB, and/or type II topoisomerase) such thateither one or both strands of the linked segments are covalently joinedat the site where the segments are linked.

[0265] A method for generating a double stranded recombinant nucleicacid molecule covalently linked in one strand can be performed bycontacting a first nucleic acid molecule which has a site-specifictopoisomerase recognition site (e.g., a type IA or a type IItopoisomerase recognition site), or a cleavage product thereof, at a 5′or 3′ terminus, with a second (or other) nucleic acid molecule, andoptionally, a topoisomerase (e.g., a type IA, type IB, and/or type IItopoisomerase), such that the second nucleotide sequence can becovalently attached to the first nucleotide sequence. As disclosedherein, the methods of the invention can be performed using any numberof nucleotide sequences, typically nucleic acid molecules wherein atleast one of the nucleotide sequences has a site-specific topoisomeraserecognition site (e.g., a type IA, type IB or type II topoisomerase), orcleavage product thereof, at one or both 5′ and/or 3′ termini.

[0266] In some embodiments, two double-stranded nucleic acid moleculescan be joined into a one larger molecule such that each strand of thelarger molecule is covalently joined (e.g., the larger molecule has nonicks). With reference to FIG. 3, a first double-stranded nucleic acidmolecule having a topoisomerase linked to each of the 5′ terminus and 3′terminus of one end may be contacted with a second nucleic acid underconditions causing the linkage of both strands of the first nucleic acidmolecule to both strands of the second nucleic acid molecule (FIG. 3A).The end of the first nucleic acid molecules to which the topoisomerasesare attached may have either a 5′-overhang, 3′-overhang or be bluntended. The end of the second nucleic acid molecule to be joined to thefirst nucleic acid molecule may have the same type of end as thetopoisomerase-linked end of the first nucleic acid molecule. The end ofthe second molecule that is not to be joined may have a different end ifdirectional joining of the segments is desired and may have the sametype of end if directionality is not required.

[0267] In another embodiment, a first nucleic acid molecule having atopoisomerase bound to the 3′ terminus of one end, and a second nucleicacid molecule having a topoisomerase bound to the 3′ terminus of one endmay be joined using the methods of the invention (FIG. 3B). A covalentlylinked double-stranded recombinant nucleic acid molecule is generated bycontacting the ends containing the topoisomerase-charged substratenucleic acid molecules.

[0268]FIG. 3C shows a first nucleic acid molecule having a topoisomerasebound to the 5′ terminus of one end, and a second nucleic acid moleculehaving a topoisomerase bound to the 5′ terminus of one end, and furthershows the production of a covalently linked double-stranded recombinantnucleic acid molecule generated by contacting the ends containing thetopoisomerase-charged substrate nucleic acid molecules.

[0269]FIG. 3D shows a nucleic acid molecule having a topoisomeraselinked to each of the 5′ terminus and 3′ terminus of both ends, andfurther shows linkage of the topoisomerase-charged nucleic acid moleculeto two nucleic acid molecules, one at each end. The topoisomerases ateach of the 5′ termini and/or at each of the 3′ termini can be the sameor different. Those skilled in the art will appreciate that nickedmolecules (e.g., covalently joined in only one strand) may be producedby omitting one of the topoisomerases from the any one of the methodsdescribed above for FIGS. 3A-3D.

[0270] A method for generating a double stranded recombinant nucleicacid molecule covalently linked in both strands can be performed, forexample, by contacting a first nucleic acid molecule having a first endand a second end, wherein, at the first end or second end or both ends,the first nucleic acid molecule has a topoisomerase recognition site (orcleavage product thereof) at or near the 5′ or 3′ terminus; at least asecond nucleic acid molecule having a first end and a second end,wherein, at the first end or second end or both ends, the at leastsecond double stranded nucleotide sequence has a topoisomeraserecognition site (or cleavage product thereof) at or near a 5′ or 3′terminus; and at least one site specific topoisomerase (e.g., a type IAand/or a type IB topoisomerase), under conditions such that allcomponents are in contact and the topoisomerase can effect its activity.A covalently linked double stranded recombinant nucleic acid generatedaccording to a method of this aspect of the invention is characterized,in part, in that it does not contain a nick in either strand at theposition where the nucleic acid molecules are joined. In one embodiment,the method is performed by contacting a first nucleic acid molecule anda second (or other) nucleic acid molecule, each of which has atopoisomerase recognition site in addition to viral sequences an/orsequences of interest, or a cleavage product thereof, at the 3′ terminior at the 5′ termini of two ends to be covalently linked. In anotherembodiment, the method is performed by contacting a first nucleic acidmolecule having a topoisomerase recognition site, or cleavage productthereof, at the 5′ terminus and the 3′ terminus of at least one end, anda second (or other) nucleic acid molecule having a 3′ hydroxyl group anda 5′ hydroxyl group at the end to be linked to the end of the firstnucleic acid molecule containing the recognition sites. As disclosedherein, the methods can be performed using any number of nucleic acidmolecules having various combinations of termini and ends.

[0271] Topoisomerases are categorized as type I, including type IA andtype IB topoisomerases, which cleave a single strand of a doublestranded nucleic acid molecule, and type II topoisomerases (gyrases),which cleave both strands of a nucleic acid molecule. Type IA and IBtopoisomerases cleave one strand of a nucleic acid molecule. Cleavage ofa nucleic acid molecule by type IA topoisomerases generates a 5′phosphate and a 3′ hydroxyl at the cleavage site, with the type IAtopoisomerase covalently binding to the 5′ terminus of a cleaved strand.In comparison, cleavage of a nucleic acid molecule by type IBtopoisomerases generates a 3′ phosphate and a 5′ hydroxyl at thecleavage site, with the type IB topoisomerase covalently binding to the3′ terminus of a cleaved strand. As disclosed herein, type I and type IItopoisomerases, as well as catalytic domains and mutant forms thereof,are useful for generating double stranded recombinant nucleic acidmolecules covalently linked in both strands according to a method of theinvention.

[0272] Type IA topoisomerases include E. coli topoisomerase I, E. colitopoisomerase III, eukaryotic topoisomerase II, archeal reverse gyrase,yeast topoisomerase III, Drosophila topoisomerase III, humantopoisomerase III, Streptococcus pneumoniae topoisomerase III, and thelike, including other type IA topoisomerases (see Berger, Biochim.Biophys. Acta 1400:3-18, 1998; DiGate and Marians, J. Biol. Chem.264:17924-17930, 1989; Kim and Wang, J. Biol. Chem. 267:17178-17185,1992; Wilson, et al., J. Biol. Chem. 275:1533-1540, 2000; Hanai, et al.,Proc. Natl. Acad. Sci., USA 93:3653-3657, 1996, U.S. Pat. No. 6,277,620,each of which is incorporated herein by reference). E. colitopoisomerase III, which is a type IA topoisomerase that recognizes,binds to and cleaves the sequence 5′-GCAACTT-3′, can be particularlyuseful in a method of the invention (Zhang, et al., J. Biol. Chem.270:23700-23705, 1995, which is incorporated herein by reference). Ahomolog, the traE protein of plasmid RP4, has been described by Li, etal., J. Biol. Chem. 272:19582-19587 (1997) and can also be used in thepractice of the invention. A DNA-protein adduct is formed with theenzyme covalently binding to the 5′-thymidine residue, with cleavageoccurring between the two thymidine residues.

[0273] Type IB topoisomerases include the nuclear type I topoisomerasespresent in all eukaryotic cells and those encoded by vaccinia and othercellular poxviruses (see Cheng, et al., Cell 92:841-850, 1998, which isincorporated herein by reference). The eukaryotic type IB topoisomerasesare exemplified by those expressed in yeast, Drosophila and mammaliancells, including human cells (see Caron and Wang, Adv. Pharmacol.29B,:271-297, 1994; Gupta, et al., Biochim. Biophys. Acta 1262:1-14,1995, each of which is incorporated herein by reference; see, also,Berger, supra, 1998). Viral type IB topoisomerases are exemplified bythose produced by the vertebrate poxviruses (vaccinia, Shope fibromavirus, ORF virus, fowlpox virus, and molluscum contagiosum virus), andthe insect poxvirus (Amsacta moorei entomopoxvirus) (see Shuman,Biochim. Biophys. Acta 1400:321-337, 1998; Petersen, et al., Virology230:197-206, 1997; Shuman and Prescott, Proc. Natl. Acad. Sci., USA84:7478-7482, 1987; Shuman, J. Biol. Chem. 269:32678-32684, 1994; U.S.Pat. No. 5,766,891; PCT/US95/16099; PCT/US98/12372, each of which isincorporated herein by reference; see, also, Cheng, et al., supra,1998).

[0274] Type II topoisomerases include, for example, bacterial gyrase,bacterial DNA topoisomerase IV, eukaryotic DNA topoisomerase II, andT-even phage encoded DNA topoisomerases (Roca and Wang, Cell 71:833-840,1992; Wang, J. Biol. Chem. 266:6659-6662, 1991, each of which isincorporated herein by reference; Berger, supra, 1998;). Like the typeIB topoisomerases, the type II topoisomerases have both cleaving andligating activities. In addition, like type IB topoisomerase, substratenucleic acid molecules can be prepared such that the type IItopoisomerase can form a covalent linkage to one strand at a cleavagesite. For example, calf thymus type II topoisomerase can cleave asubstrate nucleic acid molecule containing a 5′ recessed topoisomeraserecognition site positioned three nucleotides from the 5′ end, resultingin dissociation of the three nucleotide sequence 5′ to the cleavage siteand covalent binding the of the topoisomerase to the 5′ terminus of thenucleic acid molecule (Andersen, et al., supra, 1991). Furthermore, uponcontacting such a type II topoisomerase charged nucleic acid moleculewith a second nucleotide sequence containing a 3′ hydroxyl group, thetype II topoisomerase can ligate the sequences together, and then isreleased from the recombinant nucleic acid molecule. As such, type IItopoisomerases also are useful for performing methods of the invention.

[0275] The various topoisomerases exhibit a range of sequencespecificity. For example, type II topoisomerases can bind to a varietyof sequences, but cleave at a highly specific recognition site (seeAndersen, et al., J. Biol. Chem. 266:9203-9210, 1991, which isincorporated herein by reference.). In comparison, the type IBtopoisomerases include site specific topoisomerases, which bind to andcleave a specific nucleotide sequence (“topoisomerase recognitionsite”). Upon cleavage of a nucleic acid molecule by a topoisomerase, forexample, a type IB topoisomerase, the energy of the phosphodiester bondis conserved via the formation of a phosphotyrosyl linkage between aspecific tyrosine residue in the topoisomerase and the 3′ nucleotide ofthe topoisomerase recognition site. Where the topoisomerase cleavagesite is near the 3′ terminus of the nucleic acid molecule, thedownstream sequence (3′ to the cleavage site) can dissociate, leaving anucleic acid molecule having the topoisomerase covalently bound to thenewly generated 3′ end.

[0276] With reference to FIG. 4, a combination of restrictiondigestion/ligation and recombinational cloning may be used to constructnucleic acid molecules of the invention. A nucleic acid molecule (e.g.,a plasmid) having at least one recognition site (e.g., recombinationsite) (RSI) and at least one restriction enzyme site (RE) may beconstructed. A molecule of this type may comprise a tag sequence,optionally located adjacent to the restriction enzyme site. The moleculemay be digested with a restriction enzyme resulting in a linearmolecule. The resultant linear molecule may be contacted with a secondnucleic acid molecule comprising at least one recombination site andhaving an end compatible with the restriction digested end of the linearfirst nucleic acid molecule. In the presence of ligase and theappropriate recombination proteins, the second nucleic acid molecule iscovalently coupled to the first nucleic acid molecule replacing theportion of the first nucleic acid molecule between the recombinationsite and the restriction enzyme site. Those skilled in the art willappreciate that one or more topoisomerases may be used in place of or incombination with the restriction enzyme digestion and/or ligationreactions. Thus, the invention contemplates linear molecules, which maybe charged at one end with one or more topoisomerases, containing atleast one recombination site. The invention also contemplatescompositions comprising such molecules, reaction mixtures comprisingsuch molecules, and methods of making and using such molecules.

[0277] Suppressor tRNAs

[0278] Mutant tRNA molecules that recognize what are ordinarily stopcodons suppress the termination of translation of an mRNA molecule andare termed suppressor tRNAs. Three codons are used by both eukaryotesand prokaryotes to signal the end of gene. When transcribed into MRNA,the codons have the following sequences: UAG (amber), UGA (opal) and UAA(ochre). Under most circumstances, the cell does not contain any tRNAmolecules that recognize these codons. Thus, when a ribosome translatingan mRNA reaches one of these codons, the ribosome stalls and falls ofthe RNA, terminating translation of the mRNA. The release of theribosome from the mRNA is mediated by specific factors (see S.Mottagui-Tabar, Nucleic Acids Research 26(11), 2789, 1998). A gene withan in-frame stop codon (TAA, TAG, or TGA) will ordinarily encode aprotein with a native carboxy terminus. However, suppressor tRNAs canresult in the insertion of amino acids and continuation of translationpast stop codons.

[0279] A number of such suppressor tRNAs have been found. Examplesinclude, but are not limited to, the supE, supP, supD, supF and supZsuppressors, which suppress the termination of translation of the amberstop codon, supB, glT, supL, supN, supC and supM suppressors, whichsuppress the function of the ochre stop codon and glyT, trpT and Su-9suppressors, which suppress the function of the opal stop codon. Ingeneral, suppressor tRNAs contain one or more mutations in theanti-codon loop of the tRNA that allows the tRNA to base pair with acodon that ordinarily functions as a stop codon. The mutant tRNA ischarged with its cognate amino acid residue and the cognate amino acidresidue is inserted into the translating polypeptide when the stop codonis encountered. For a more detailed discussion of suppressor tRNAs, thereader may consult Eggertsson, et al., (1988) Microbiological Review52(3):354-374, and Engleerg-Kukla, et al. (1996) in Escherichia coli andSalmonella Cellular and Molecular Biology, Chapter 60, pps 909-921,Neidhardt, et al. eds., ASM Press, Washington, D.C.

[0280] Mutations that enhance the efficiency of termination suppressors,i.e., increase the read through of the stop codon, have been identified.These include, but are not limited to, mutations in the uar gene (alsoknown as the prfA gene), mutations in the ups gene, mutations in thesueA, sueB and sueC genes, mutations in the rpsD (ramA) and rpsE (spcA)genes and mutations in the rplL gene.

[0281] Under ordinary circumstances, host cells would not be expected tobe healthy if suppression of stop codons is too efficient. This isbecause of the thousands or tens of thousands of genes in a genome, asignificant fraction will naturally have one of the three stop codons;complete read-through of these would result in a large number ofaberrant proteins containing additional amino acids at their carboxytermini. If some level of suppressing tRNA is present, there is a racebetween the incorporation of the amino acid and the release of theribosome. Higher levels of tRNA may lead to more read-through althoughother factors, such as the codon context, can influence the efficiencyof suppression.

[0282] Organisms ordinarily have multiple genes for tRNAs. Combined withthe redundancy of the genetic code (multiple codons for many of theamino acids), mutation of one tRNA gene to a suppressor tRNA status doesnot lead to high levels of suppression. The TAA stop codon is thestrongest, and most difficult to suppress. The TGA is the weakest, andnaturally (in E. coli) leaks to the extent of 3%. The TAG (amber) codonis relatively tight, with a read-through of ˜1% without suppression. Inaddition, the amber codon can be suppressed with efficiencies on theorder of 50% with naturally occurring suppressor mutants. Suppression insome organisms (e.g., E. coli) may be enhanced when the nucleotidefollowing the stop codon is an adenosine. Thus, the present inventioncontemplates nucleic acid molecules having a stop codon followed by anadenosine (e.g., having the sequence TAGA, TAAA, and/or TGAA).

[0283] Suppression has been studied for decades in bacteria andbacteriophages. In addition, suppression is known in yeast, flies,plants and other eukaryotic cells including mammalian cells. Forexample, Capone, et al. (Molecular and Cellular Biology 6(9):3059-3067,1986) demonstrated that suppressor tRNAs derived from mammalian tRNAscould be used to suppress a stop codon in mammalian cells. A copy of theE. coli chloramphenicol acetyltransferase (cat) gene having a stop codonin place of the codon for serine 27 was transfected into mammalian cellsalong with a gene encoding a human serine tRNA that had been mutated toform an amber, ochre, or opal suppressor derivative of the gene.Successful expression of the cat gene was observed. An induciblemammalian amber suppressor has been used to suppress a mutation in thereplicase gene of polio virus and cell lines expressing the suppressorwere successfully used to propagate the mutated virus (Sedivy, et al.,Cell 50: 379-389 (1987)). The context effects on the efficiency ofsuppression of stop codons by suppressor tRNAs has been shown to bedifferent in mammalian cells as compared to E. coli (Phillips-Jones, etal., Molecular and Cellular Biology 15(12): 6593-6600 (1995), Martin, etal., Biochemical Society Transactions 21: (1993)) Since some humandiseases are caused by nonsense mutations in essential genes, thepotential of suppression for gene therapy has long been recognized (seeTemple, et al., Nature 296(5857):537-40 (1982)). The suppression ofsingle and double nonsense mutations introduced into the diphtheriatoxin A-gene has been used as the basis of a binary system for toxingene therapy (Robinson, et al., Human Gene Therapy 6:137-143 (1995)).

[0284] Use of Suppressor tRNAs to Conditionally Express Fusion Proteins

[0285] Because the methods used to create the nucleic acids of theinvention are site specific, the orientation and/or reading frame of anucleic acid sequence on a first nucleic acid molecule can be controlledwith respect to the orientation and/or reading frame of a sequence on asecond nucleic acid molecule when all or a portion of the molecules arejoined in a recombination and/or topoisomerase-mediated reaction. Thiscontrol makes the construction of fusions between sequences present ondifferent nucleic acid molecules a simple matter.

[0286] In general terms, an open reading frame may be expressed in fourforms: native at both amino and carboxy termini, modified at either end,or modified at both ends. A nucleic acid sequence of interest comprisingan ORF of interest may include the N-terminal methionine ATG codon, anda stop codon at the carboxy end, of the ORF, thus ATG-ORF-stop.Frequently, the nucleic acid molecule comprising the sequence ofinterest will include translation initiation sequences, tis, that may belocated upstream of the ATG that allow expression of the gene, thustis-ATG-ORF-stop. Constructs of this sort allow expression of an ORF asa protein that contains the same amino and carboxy amino acids as in thenative, uncloned, protein. When such a construct is fused in-frame withan amino-terminal protein tag, e.g., GST, the tag will have its own tis,thus tis-ATG-tag-tis-ATG-ORF-stop, and the bases comprising the tis ofthe ORF will be translated into amino acids between the tag and the ORF.In addition, some level of translation initiation may be expected in theinterior of the mRNA (i.e., at the ORF's ATG and not the tag's ATG)resulting in a certain amount of native protein expression contaminatingthe desired protein.

[0287] DNA (lower case): tis1-atg-tag-tis2-atg-orf-stop

[0288] RNA (lower case, italics): tis1-atg-tag-tis2-atg-orf-stop

[0289] Protein (upper case): ATG-TAG-TIS2-ATG-ORF (tis1 and stop are nottranslated)+contaminating ATG-ORF (translation of ORF beginning attis2).

[0290] Using the methods disclosed herein, one skilled in the art canconstruct a vector containing a tag adjacent to a recombination sitepermitting the in frame fusion of a tag to the C- and/or N-terminus ofthe ORF of interest.

[0291] Given the ability to rapidly create a number of clones in avariety of vectors, there is a need in the art to maximize the number ofways a single cloned ORF can be expressed without the need to manipulatethe construct itself. The present invention meets this need by providingmaterials and methods for the controlled expression of a C- and/orN-terminal fusion to a target ORF using one or more suppressor tRNAs tosuppress the termination of translation at a stop codon. Thus, thepresent invention provides materials and methods in which a geneconstruct is prepared flanked with recombination sites.

[0292] The construct may be prepared with a sequence coding for a stopcodon preferably at the C-terminus of the ORF encoding the protein ofinterest. In some embodiments, a stop codon can be located adjacent tothe ORF, for example, within the recombination site flanking the gene orat or near the 3′ end of the sequence of interest before a recombinationsite. The target gene construct can be transferred through recombinationto various vectors that can provide various C-terminal or N-terminaltags (e.g., GFP, GST, His Tag, GUS, etc.) to the ORF of interest. Whenthe stop codon is located at the carboxy terminus of the ORF, expressionof the ORF with a “native” carboxy end amino acid sequence occurs undernon-suppressing conditions (i.e., when the suppressor tRNA is notexpressed) while expression of the ORF as a carboxy fusion proteinoccurs under suppressing conditions. Those skilled in the art willrecognize that any suppressors and any codons could be used in thepractice of the present invention. Suppressors may insert any amino acidat the position corresponding to the stop codon, for example, Ala, Arg,Asn, Asp, Cys, Gln, Glu, Gly, His, Ile, Leu, Lys, Met, Phe, Pro, Ser,Thr, Trp, Tyr, or Val may be inserted. In some embodiments, serine maybe inserted.

[0293] In some embodiments, the gene coding for the suppressing tRNA maybe incorporated into the vector from which the target ORF is to beexpressed. In other embodiments, the gene for the suppressor tRNA may bein the genome of the host cell. In still other embodiments, the gene forthe suppressor may be located on a separate viral vector or othervector—i.e., plasmid—and provided in trans. In embodiments of this type,the vector containing the suppressor gene may be a recombinantadenoviral vector and cells may be co-infected with a viral vectorexpressing a sequence of interest and a viral vector expressing asuppressor tRNA.

[0294] More than one copy of a suppressor tRNA may be provided in all ofthe embodiments described herein. For example, a host cell may beprovided that contains multiple copies of a gene encoding the suppressortRNA. Alternatively, multiple gene copies of the suppressor tRNA underthe same or different promoters may be provided in the same vectorbackground as the target ORF of interest. In some embodiments, multiplecopies of a suppressor tRNA may be provided in a different vector thanthe one containing the target ORF of interest. In other embodiments, oneor more copies of the suppressor tRNA gene may be provided on the vectorcontaining the ORF for the protein of interest and/or on another vectorand/or in the genome of the host cell or in combinations of the above.When more than one copy of a suppressor tRNA gene is provided, the genesmay be expressed from the same or different promoters that may be thesame or different as the promoter used to express the ORF encoding theprotein of interest.

[0295] In some embodiments, two or more different suppressor tRNA genesmay be provided. In embodiments of this type one or more of theindividual suppressors may be provided in multiple copies and the numberof copies of a particular suppressor tRNA gene may be the same ordifferent as the number of copies of another suppressor tRNA gene. Eachsuppressor tRNA gene, independently of any other suppressor tRNA gene,may be provided on the vector used to express the ORF of interest and/oron a different vector and/or in the genome of the host cell. A giventRNA gene may be provided in more than one place in some embodiments.For example, a copy of the suppressor tRNA may be provided on the vectorcontaining the ORF of interest while one or more additional copies maybe provided on an additional vector and/or in the genome of the hostcell. When more than one copy of a suppressor tRNA gene is provided, thegenes may be expressed from the same or different promoters that may bethe same or different as the promoter used to express the ORF encodingthe protein of interest and may be the same or different as a promoterused to express a different tRNA gene.

[0296] In some embodiments of the present invention, the target ORF ofinterest and the gene expressing the suppressor tRNA may be controlledby the same promoter. In other embodiments, the target ORF of interestmay be expressed from a different promoter than the suppressor tRNA.Those skilled in the art will appreciate that, under certaincircumstances, it may be desirable to control the expression of thesuppressor tRNA and/or the target ORF of interest using a regulatablepromoter. For example, either the target ORF of interest and/or the geneexpressing the suppressor tRNA may be controlled by a promoter such asthe lac promoter or derivatives thereof such as the tac promoter. Insome embodiments, both the target ORF of interest and the suppressortRNA gene are expressed from the T7 RNA polymerase promoter and,optionally, are expressed as part of one RNA molecule. In embodiments ofthis type, the portion of the RNA corresponding to the suppressor tRNAis processed from the originally transcribed RNA molecule by cellularfactors.

[0297] In some embodiments, the expression of the suppressor tRNA genemay be under the control of a different promoter from that of the ORF ofinterest. In some embodiments, it may be possible to express thesuppressor gene before the expression of the target ORF. This wouldallow levels of suppressor to build up to a high level, before they areneeded to allow expression of a fusion protein by suppression of a thestop codon. For example, in embodiments of the invention where thesuppressor gene is controlled by a promoter inducible with IPTG, thetarget ORF is controlled by the T7 RNA polymerase promoter and theexpression of the T7 RNA polymerase is controlled by a promoterinducible with an inducing signal other than IPTG, e.g., NaCl, one couldturn on expression of the suppressor tRNA gene with IPTG prior to theinduction of the T7 RNA polymerase gene and subsequent expression of theORF of interest. In some embodiments, the expression of the suppressortRNA might be induced about 15 minutes to about one hour before theinduction of the T7 RNA polymerase gene. In one embodiment, theexpression of the suppressor tRNA may be induced from about 15 minutesto about 30 minutes before induction of the T7 RNA polymerase gene. Insome embodiments, the expression of the T7 RNA polymerase gene is underthe control of an inducible promoter.

[0298] In additional embodiments, the expression of the target ORF ofinterest and the suppressor tRNA can be arranged in the form of afeedback loop. For example, the target ORF of interest may be placedunder the control of the T7 RNA polymerase promoter while the suppressorgene is under the control of both the T7 promoter and the lac promoter.The T7 RNA polymerase gene itself is also under the control of both theT7 promoter and the lac promoter. In addition, the T7 RNA polymerasegene has an amber stop mutation replacing a normal tyrosine codon, e.g.,the 28th codon (out of 883). No active T7 RNA polymerase can be madebefore levels of suppressor are high enough to give significantsuppression. Then expression of the polymerase rapidly rises, becausethe T7 polymerase expresses the suppressor gene as well as itself. Inother preferred embodiments, only the suppressor gene is expressed fromthe T7 RNA polymerase promoter. Embodiments of this type would give ahigh level of suppressor without producing an excess amount of T7 RNApolymerase. In other preferred embodiments, the T7 RNA polymerase genehas more than one amber stop mutation. This will require higher levelsof suppressor before active T7 RNA polymerase is produced.

[0299] In some embodiments of the present invention it may be desirableto have more than one stop codon suppressible by more than onesuppressor tRNA. A recombinant viral vector may be constructed so as topermit the regulatable expression of N- and/or C-terminal fusions of aprotein of interest from the same construct. A viral vector may comprisea first tag sequence expressed from a promoter and may include a firststop codon in the same reading frame as the tag. The stop codon may belocated anywhere in the tag sequence and is preferably located at ornear the C-terminal of the tag sequence. The stop codon may also belocated in a recombination site or in an internal ribosome entrysequence (IRES). The viral vector may also include a sequence ofinterest preferably comprising a ORF of interest that includes a secondstop codon. The first tag and the ORF of interest are preferably in thesame reading frame although inclusion of a sequence that causes frameshifting to bring the first tag into the same reading frame as the ORFof interest is within the scope of the present invention. The secondstop codon is preferably in the same reading frame as the ORF ofinterest and is preferably located at or near the end of the codingsequence for the ORF. The second stop codon may optionally be locatedwithin a recombination site located 3′ to the sequence of interest. Theconstruct may also include a second tag sequence in the same readingframe as the ORF of interest and the second tag sequence may optionallyinclude a third stop codon in the same reading frame as the second tag.A transcription terminator and/or a polyadenylation sequence may beincluded in the construct after the coding sequence of the second tag.The first, second and third stop codons may be the same or different. Insome embodiments, all three stop codons are different. In embodimentswhere the first and the second stop codons are different, the sameconstruct may be used to express an N-terminal fusion, a C-terminalfusion and the native protein by varying the expression of theappropriate suppressor tRNA. For example, to express the native protein,no suppressor tRNAs are expressed and protein translation is controlledby an appropriately located IRES. When an N-terminal fusion is desired,a suppressor tRNA that suppresses the first stop codon is expressedwhile a suppressor tRNA that suppresses the second stop codon isexpressed in order to produce a C-terminal fusion. In some instances itmay be desirable to express a doubly tagged protein of interest in whichcase suppressor tRNAs that suppress both the first and the second stopcodons may be expressed.

[0300] Construction and Uses Nucleic Acid Molecules of the Invention.

[0301] As discussed below in more detail, in one aspect, the inventionprovides a modular system for constructing viruses, e.g., viral vectors,having particular functions or activities. The present invention alsoincludes methods for preparing viruses, e.g., viral vectors, containingmore than one nucleic acid insert (e.g., two, three, four, five, six,eight, ten, twelve, fifteen, twenty, thirty, forty, fifty, etc.inserts). In one general embodiment of the invention, viral vectorsand/or nucleic acids molecules of the invention are prepared as follows.Nucleic acid molecules that are to ultimately be incorporated into theviral vector are obtained (e.g., purchased, prepared by PCR or by thepreparation of cDNA using reverse transcriptase). Suitable recombinationsites are either incorporated into the 5′ and/or 3′ ends of the nucleicacid molecules during synthesis or added later. A nucleic acidcomprising all or a portion of a viral genome and the nucleic acid to beincorporated are combined in the presence of one or more recombinationproteins in order to construct the desired viral vector.

[0302] In some embodiments of the invention nucleic acid molecules ofthe invention may be combined using various combinations of techniquesknown in the art. When a first nucleic acid molecule is to be joinedwith a second nucleic acid molecule, the ends of the molecules may bejoined using the same or different techniques. For example, one end of afirst nucleic acid molecule to be joined with a second nucleic acidmolecule may comprise one type of recognition site (e.g., atopoisomerase site) and the other end may comprise a different type ofsite (e.g., a recombination site or a restriction enzyme site). Invarious embodiments, a nucleic acid molecule may have a restrictionenzyme site on one end and a topoisomerase site on the other end, arestriction enzyme site on one end and a recombination site on the otherend, or a topoisomerase site on one end and a recombination site on theother end. Those skilled in the art will appreciate that a ligase and/ortopoisomerase may be used to link an end having a restriction site withanother nucleic acid molecule. When topoisomerase is used to join twonucleic acid molecules, either or both strands may be covalently joined.FIG. 3 shows examples of the covalent joining of both strands.

[0303] To construct a modular viral vector, one or more nucleic acidsegments comprising one or more recombination sites and also comprisinga viral sequence may be prepared. In some embodiments, multiplesegments, each having at least one recombination site and some havingviral sequences (e.g., baculoviral or adenoviral sequences) may beconstructed and combined to produce a nucleic acid molecule of theinvention. For example, a nucleic acid segment comprising an adenoviralITR and a recombination site may be prepared. Further, a plurality ofnucleic acid segments, each comprising a different portion of theadenoviral genome flanked by recombination sites, may be prepared. Insome embodiments, the entire genome of an adenovirus is prepared insegments flanked by recombination sites. Such segments may be combinedwith one or more additional segments comprising additional sequences ofinterest such that, after combining, a nucleic acid comprising all or aportion of an adenoviral genome and comprising a sequence of interest isformed.

[0304] Segments of an adenoviral genome may be prepared from differentserotypes of adenovirus, for example, Ad5, Ad3, Ad10, etc., and viralvectors having a mixed serotype, (e.g., some determinants of Ad5 andsome of Ad10) may be prepared. It may be desirable to vary the mostimmunogenic portions of the viruses in situations where multipleadministrations of viral vectors are contemplated.

[0305] Each segment of the adenoviral genome may comprise one or moreregions of the genome, for example, left ITR, right ITR, packagingsignal, E1, E2, E3, E4, and/or one or more late regions. In someembodiments, a segment may comprise the entire adenoviral genome exceptone region that is on a different segment. For example, an entireadenoviral genome except for the packaging signal may be prepared on onesegment and the packaging signal may be prepared on a different segment.The two segments may be combined (e.g., using recombinational cloning)to produce a viral vector of the invention. Likewise, an entireadenoviral genome may be prepared that lacks one or more of thefollowing elements: left ITR, E1, E2, E3, E4, or right ITR. The lackingelement may be prepared on a separate segment and the two segments maybe combined to produce a viral vector. One or more sequences of interestmay be incorporated into either segment prior to combining the segmentsin order to produce an adenoviral vector containing one or moresequences of interest. More than one viral region may be prepared on asegment, for example, the left ITR, packaging signal, and E3 region maybe prepared on one segment with the remainder of the adenoviralfunctions necessary to prepare a viral vector present on one or moreother segments. Sequences of interest may be present on any one of thesegments.

[0306] Typically, the nucleic acid molecules may be dissolved in anaqueous buffer and added to the reaction mixture. One suitable set ofconditions is 4 μl CLONASE™ enzyme mixture (e.g., InvitrogenCorporation, Cat. Nos. 11791-019 and 11789-013), 4 μl 5× reaction bufferand nucleic acid and water to a final volume of 20 μl. This willtypically result in the inclusion of about 200 ng of Int and about 80 ngof IHF in a 20 μl BP reaction and about 150 ng Int, about 25 ng IHF andabout 30 ng Xis in a 20 μl LR reaction.

[0307] Proteins for conducting an LR reaction may be stored in asuitable buffer, for example, LR Storage Buffer, which may compriseabout 50 mM Tris at about pH 7.5, about 50 mM NaCl, about 0.25 mM EDTA,about 2.5 mM Spermidine, and about 0.2 mg/ml BSA. When stored, proteinsfor an LR reaction may be stored at a concentration of about 37.5 ng/μlINT, 10 ng/μl IHF and 15 ng/μl XIS. Proteins for conducting a BPreaction may be stored in a suitable buffer, for example, BP StorageBuffer, which may comprise about 25 mM Tris at about pH 7.5, about 22 mMNaCl, about 5 mM EDTA, about 5 mM Spermidine, about 1 mg/ml BSA, andabout 0.0025% Triton X-100. When stored, proteins for an BP reaction maybe stored at a concentration of about 37.5 ng/μl INT and 20 ng/μl IHF.One skilled in the art will recognize that enzymatic activity may varyin different preparations of enzymes. The amounts suggested above may bemodified to adjust for the amount of activity in any specificpreparation of enzymes.

[0308] A suitable 5×reaction buffer for conducting recombinationreactions may comprise 100 mM Tris pH 7.5, 88 mM NaCl, 20 mM EDTA, 20 mMSpermidine, and 4 mg/ml BSA. Thus, in a recombination reaction, thefinal buffer concentrations may be 20 mM Tris pH 7.5, 17.6 mM NaCl, 4 mMEDTA, 4 mM Spermidine, and 0.8 mg/ml BSA. Those skilled in the art willappreciate that the final reaction mixture may incorporate additionalcomponents added with the reagents used to prepare the mixture, forexample, a BP reaction may include 0.005% Triton X-100 incorporated fromthe BP Clonase™.

[0309] In some preferred embodiments, particularly those in which attLsites are to be recombined with attR sites, the final reaction mixturemay include about 50 mM Tris HCl, pH 7.5, about 1 mM EDTA, about 1 mg/mlBSA, about 75 mM NaCl and about 7.5 mM spermidine in addition torecombination enzymes and the nucleic acids to be combined. In otherpreferred embodiments, particularly those in which an attB site is to berecombined with an attP site, the final reaction mixture may includeabout 25 mM Tris HCl, pH 7.5, about 5 mM EDTA, about 1 mg/ml bovineserum albumin (BSA), about 22 mM NaCl, and about 5 mM spermidine.

[0310] In some preferred embodiments, particularly those in which attLsites are to be recombined with attR sites, the final reaction mixturemay include about 40 mM Tris HCl, pH 7.5, about 1 mM EDTA, about 1 mg/mlBSA, about 64 mM NaCl and about 8 mM spermidine in addition torecombination enzymes and the nucleic acids to be combined. One of skillin the art will appreciate that the reaction conditions may be variedsomewhat without departing from the invention. For example, the pH ofthe reaction may be varied from about 7.0 to about 8.0; theconcentration of buffer may be varied from about 25 mM to about 100 mM;the concentration of EDTA may be varied from about 0.5 mM to about 2 mM;the concentration of NaCl may be varied from about 25 mM to about 150mM; and the concentration of BSA may be varied from 0.5 mg/ml to about 5mg/ml. In other preferred embodiments, particularly those in which anattB site is to be recombined with an attP site, the final reactionmixture may include about 25 mM Tris HCl, pH 7.5, about 5 mM EDTA, about1 mg/ml bovine serum albumin (BSA), about 22 mM NaCl, about 5 mMspermidine and about 0.005% detergent (e.g., Triton X-100).

[0311] The invention also includes viral vectors, in addition toadenoviral vectors (e.g., baculoviral vectors), which contain either allor, part of one or more viral genome. Using vectors comprisingbaculoviral nucleic acid for purposes of illustration, vectors of theinvention include those which comprise one or more element (e.g., one ormore functional element) of a baculoviral genome, as well as vectorswhich comprise one or more element (e.g., promoters, transcriptionterminators, polyA signals or sequences, ribosome binding sites,enhancers, ORFs or portions thereof, etc.) of one or more other viralgenomes. Typically, these vectors will include one or more recombinationsite, as described elsewhere herein.

[0312] One specific example of a vector of the invention is shownschematically in FIG. 13. This vector contains three separatebaculoviral elements. More specifically, the vector shown in FIG. 13comprises the IE2 gene promoter and IE2 gene polyA region of Orgyiapseudotsugata. The vector also includes the GP64 promoter of Autographacalifornica. Thus, nucleic acid molecules of the invention includevectors which contain one or more elements (e.g., an element describedherein) derived from one or more viral genome (e.g., adenoviral genome,baculoviral genome, etc.). Further, these elements may be from the sameor different viruses.

[0313] The invention further includes nucleic acid molecules whichcomprise modified elements of viral genomes. These modified elements maybe defined and/or described within the scope of the invention in anynumber of ways. Examples of such ways include (1) function (e.g., aproperty conferred upon a nucleic acid which contains the element), (2)% sequence identity, and (3) % homology or sequence identity ofexpression products, as well as combinations of these ways. Percenthomology or sequence identity will typically be determined withreference to the nucleotide or amino acid sequence of another nucleicacid or polypeptide.

[0314] As indicated above, viral elements and modified viral elementssuitable for use with the invention may be described by their ability toconfer one or more functional properties on nucleic acid molecules whichcontain them. Using the GP64 promoter as an example, this promoter is aninducible promoter which exhibits low level basal constitutive activity.In other words, in the absence of induction, the GP64 promoter allowsfor low level of transcription when operably linked to a nucleic acidsegment. Functional properties are also associated with other viralelements, such as origins of replication, polyA tail sequences,packaging signals, LTRs, etc. Further, depending on the particularelement, functional activity can be assessed at either the level of thevector (e.g., the DNA or RNA level), a transcription product (e.g., theRNA level), and/or a translation product (e.g., the polypeptide level).Thus, the invention further includes nucleic acid molecules whichcomprise modified viral elements which retain all or some of thefunctions of the viral elements from which they are derived (e.g., the“wild-type” viral element). In many instances, a modified element willretain at least one functional property of the element from which theyare derived. In particular embodiments, the modified element will (1)have at least one additional property not associated with the elementfrom which it was derived, (2) be deficient in at least one propertyassociated with the element from which it was derived, and/or (3) haveincreased or decreased activity with respect to at least one propertyassociated with the element from which it was derived.

[0315] As also indicated above, modified elements (e.g., modified viralelements) contained in nucleic acid molecules of the invention may bedescribed by their structural similarity to elements from which they arederived. For example, modified elements may be at least 50% identical,at least 55% identical, at least 60% identical, at least 65% identical,at least 70% identical, at least 75% identical, at least 80% identical,at least 85% identical, at least 90% identical, or at least 95%identical at the nucleic acid level to the nucleic acid molecules fromwhich they are derived. Modified elements may also be defined by havingsufficient structural similarity to the nucleic acid molecules fromwhich they are derived (e.g., an element the nucleotide sequence ofwhich is set out elsewhere herein) so that the two nucleic acids willhybridized. Often, these molecules will hybridized to each other understringent hybridization conditions. In many instances, these modifiedelements will retain at least one property associated from the elementsfrom which they are derived.

[0316] When modified elements of a viral genome encode a polypeptideexpression product, the polypeptide may be at least 50% identical orhomologous, at least 55% identical or homologous, at least 60% identicalor homologous, at least 65% identical or homologous, at least 70%identical or homologous, at least 75% identical or homologous, at least80% identical or homologous, at least 85% identical or homologous, atleast 90% identical or homologous, or at least 95% identical orhomologous at the amino acid level to the amino acid sequences of thepolypeptide which is expressed from the nucleic acid from which themodified elements is derived. Typically, polypeptide expression productsof modified elements will retain at least one functional property ofpolypeptides which are expressed from nucleic acids from which themodified elements are derived. In particular embodiments, thepolypeptide expression product of a modified element will (1) have atleast one additional property not associated with the polypeptideexpression product from which the element from which it was derived, (2)be deficient in at least one property associated with the polypeptideexpression product from which the element from which it was derived,and/or (3) have increased or decreased activity with respect to at leastone property associated with the polypeptide expression product fromwhich the element from which it was derived.

[0317] One example of a vector of the invention is a vector whichcontains the GP64 promoter of Autographa californica operably linked toa heterologous nucleic acid. In particular embodiments, the GP64promoter has all or part of the nucleotide sequence set out in Table 12beginning at nucleotide 3364. The invention further include nucleic acidmolecules which comprise modified forms of the GP64 promoter. Thesemodified forms of the GP64 promoter include deleted forms of thepromoter which comprise at least 30 nucleotides, at least 40nucleotides, at least 50 nucleotides, at least 60 nucleotides, at least70 nucleotides, at least 80 nucleotides, at least 90 nucleotides, or atleast 95 nucleotides.

[0318] As indicated above, vectors of the invention may comprise all orpart of a viral genome. For example, vectors of the invention maycomprise at least about 5%, at least about 10%, at least about 20%, atleast about 30%, at least about 40%, at least about 50%, at least about60%, at least about 70%, at least about 80%, at least about 90%, atleast about 95%, or 100% of a viral genome used to prepare the vector.For example, a baculoviral vector which contains about 50% of the usedto prepare it may contain about 66 kb of baculoviral nucleic acid.

[0319] It is not necessary that all viral functions required forreplication be contained on a segment and be included in the finalnucleic acid molecule comprising all or a portion of the viral genome.One or more required functions may be provided in trans. For example, arequired function may be incorporated into the genome of a cell line andstill provide the function.

[0320] Viruses lacking the function could be prepared in the cell lineexpressing the function. These viruses could only replicate in the cellline expressing the function and, thus, would be replication-deficientin any other cell line. Any required function could be used in thisfashion, for example, the adenovirus E2 and/or E4 functions (see,Weinberg, et al., Proc. Ntl. Acad. Sci. USA 80:5383, 5386, 1983).

[0321] Segments prepared as above may be linear fragments (e.g., PCRfragments) or segments may be part of larger nucleic acid molecule(e.g., a plasmid). The segments may be combined to form a viral vectorof the invention. When the segments are combined, the resultantadenoviral vector may be a linear molecule, for example, by combininglinear segments using recombination cloning. A linear viral vector maybe introduced (e.g., by transfection, electroporation, etc.) into anappropriate host cell and packaged virus may be isolated as describedelsewhere herein. Alternatively, a viral vector may be prepared as partof a circular molecule (e.g., a plasmid) and the viral vector mayreleased from the circular molecule (e.g., by restriction digest) andintroduced into an appropriate host cell and packaged virus isolated.

[0322] When one seeks to prepare or construct a viral vector containingmultiple nucleic acid inserts, these inserts can be inserted into aviral vector in either one reaction mixture or a series of reactionmixtures. For example, multiple nucleic acid segments can be linked endto end and inserted into a viral vector using reactions performed, forexample, in a single reaction mixture. The nucleic acid segments in thisreaction mixture can be designed so that recombination sites on their 5′and 3′ ends result in their insertion into a nucleic acid comprising allor a portion of a viral genome in a specific order and a specific 5′ to3′ orientation. Alternatively, nucleic acid segments can be designed sothat they are inserted into a nucleic acid comprising all or a portionof a viral genome without regard to order, orientation (i.e., 5′ to 3′orientation), the number of inserts, and/or the number of duplicateinserts.

[0323] Methods of the invention can also be used to prepare viralvectors that, upon expression of a sequence of interest contained in theviral vector, produce one or more polypeptides having one or moredesired property, function, or activity (e.g., an enzymatic activity,the ability to bind a nucleic acid, etc.). For example, a polypeptidehaving one or more enzymatic activities might be expressed from theviral vectors of the present invention. Viral vectors of this type mightbe used, for example, in a gene therapy protocol to replace a missingenzymatic activity. Polypeptides produced from the viral vectors of thepresent invention may have other desirable characteristics, for example,a polypeptide may comprise one or more antigenic determinants.Expression of such a polypeptide may result in an immune responsespecific for the expressed polypeptide. Such a viral vector may be used,for example, as an immunotherapeutic, for example, a vaccine.

[0324] Methods of the invention can also be used to prepare viralvectors that, upon expression of a sequence of interest contained in theviral vector, produce one or more un-translated RNA molecules, forexample, ribozymes, antisense molecules, RNAi and the like. Such a viralvector might be used, for example, to modulate (e.g., inhibit) theexpression of one more RNA or polypeptide molecules produced by a hostorganism. Such a vector might be used, for example, to inhibit theexpression of a disease associated RNA or polypeptide.

[0325] Methods of the invention can also be used to prepare viralvectors that, upon expression of a sequence of interest contained in theviral vector, produce fusion proteins having more than one property,function, or activity. Further, the expression product can be producedin such a manner as to facilitate its export from the cell. For example,these expression products can be fusion proteins that contain a signalpeptide that results in export of the protein from the cell. Oneapplication where cell export may be desirable is where the proteinsthat are to be exported are enzymes that interact with extracellularsubstrates.

[0326] In a specific embodiment, the invention further provides methodsfor introducing viral vectors and/or nucleic acids molecules of theinvention into animals (e.g., humans) and animal cells (e.g., humancells), as part of a gene therapy protocol. Viral vectors of the presentinvention may be designed such that compositions comprising the vectorsare free of viral vectors that are replication competent in the targetcell. Thus, in some embodiments, viral vectors of the present inventionare replication restricted, i.e., can replicate in a permissive celltype, e.g., 293 cells, and cannot replicate in a target cell type, e.g.,patient cells.

[0327] Gene therapy refers to therapy performed by the administration toa subject of an expressed or expressible nucleic acid molecule. In manyembodiments of the invention, nucleic acid molecules of the inventionwill encoded one or more proteins (e.g., one or more fusion proteins)that mediate at least one therapeutic effect. Thus, the inventionprovide nucleic acid molecules and methods for use in gene therapy.

[0328] Viral vectors and/or nucleic acids molecules of the invention canbe used to prepare gene therapy vectors designed to replace genes thatreside in the genome of a cell, to delete such genes, or to insert aheterologous gene or groups of genes. When viral vectors and/or nucleicacids molecules of the invention function to delete or replace a gene orgenes, the gene or genes being deleted or replaced may lead to theexpression of either a “normal” phenotype or an aberrant phenotype. Oneexample of an aberrant phenotype is the disease cystic fibrosis.Further, the gene therapy vectors may be either stably maintained (e.g.,integrate into cellular nucleic acid by homologous or site specificrecombination) or non-stably maintained in cells.

[0329] Further, viral vectors and/or nucleic acids molecules of theinvention may be used to suppress “abnormal” phenotypes or complement orsupplement “normal” phenotypes that result from the expression ofendogenous genes. One example of a viral vector of the inventiondesigned to suppress an abnormal phenotype would be where an expressionproduct of the viral vector has dominant/negative activity. An exampleof a viral vector of the invention designed to supplement a normalphenotype would be where introduction of the viral vector effectivelyresults in the amplification of a gene resident in the cell.

[0330] In some embodiments, viral vectors and/or nucleic acids of thepresent invention may be used to prevent or inhibit the expression ofone or more genes in an organism, for example, by homology-dependentgene silencing (HDGS, see, for example, Bernstein, et al., RNA 7:1509-21(2001), and Bass, Cell 101:235-238 (2000)). The genes expression ofwhich is to be inhibited, i.e., silenced, may be endogenous to theorganism or may be exogenous to the organism.

[0331] Viral vectors and/or nucleic acid molecules of the invention maybe prepared to generate interfering RNAs (RNAi). RNAi is double-strandedRNA that results in degradation of specific mRNAs, and can also be usedto lower or eliminate gene expression. Viral vectors and/or nucleic acidmolecules of the invention may be engineered, for example, to producedsRNA molecules by, for example, engineering the viral vectors and/ornucleic acid molecules to have a sequence that, when transcribed, foldsback upon itself to generate a hairpin molecule containing adouble-stranded portion. One strand of the double-stranded portion maycorrespond to all or a portion of the sense strand of the mRNAtranscribed from the gene to be silenced while the other strand of thedouble-stranded portion may correspond to all or a portion of theantisense strand. Other methods of producing a double-stranded RNAmolecule may be used, for example, a viral vector and/or nucleic acidmolecules may be engineered to have a first sequence that, whentranscribed, corresponds to all or a portion of the sense strand of theMRNA transcribed from the gene to be silenced and a second sequencethat, when transcribed, corresponds to all or portion of an antisensestrand (i.e., the reverse complement) of the mRNA transcribed from thegene to be silenced. This may be accomplished by putting the first andthe second sequence on the same strand of the viral vector each underthe control of its own promoter. Alternatively, two promoters may bepositioned on opposite strands of the viral vector such that expressionfrom each promoter results in transcription of one strand of thedouble-stranded RNA. In some embodiments, it may be desirable to havethe first sequence on one viral vector or nucleic acid molecule and thesecond sequence on a second viral vector or nucleic acid molecule and tointroduce both vectors or molecules into a cell containing the gene tobe silenced. In other embodiments, a viral vector or nucleic acidmolecule containing only the antisense strand may be introduced and themRNA transcribed from the gene to be silenced may serve as the otherstrand of the double-stranded RNA. In some embodiments, a dsRNA to beused to silence a gene may have one or more regions of homology to agene to be silenced. Regions of homology may be from about 20 bp toabout 5 kbp in length, 20 bp to about 4 kbp in length, 20 bp to about 3kbp in length, 20 bp to about 2.5 kbp in length, from about 20 bp toabout 2 kbp in length, 20 bp to about 1.5 kbp in length, from about 20bp to about 1 kbp in length, 20 bp to about 750 bp in length, from about20 bp to about 500 bp in length, 20 bp to about 400 bp in length, 20 bpto about 300 bp in length, 20 bp to about 250 bp in length, from about20 bp to about 200 bp in length, from about 20 bp to about 150 bp inlength, from about 20 bp to about 100 bp in length, from about 20 bp toabout 90 bp in length, from about 20 bp to about 80 bp in length, fromabout 20 bp to about 70 bp in length, from about 20 bp to about 60 bp inlength, from about 20 bp to about 50 bp in length, from about 20 bp toabout 40 bp in length, from about 20 bp to about 30 bp in length, fromabout 20 bp to about 25 bp in length, from about 15 bp to about 25 bp inlength, from about 17 bp to about 25 bp in length, from about 19 bp toabout 25 bp in length, from about 15 bp to about 23 bp, from about 17 bpto about 23 bp, from about 19 bp to about 23 bp in length, from about 15bp to about 21 bp, from about 17 bp to about 21 bp, or from about 19 bpto about 21 bp in length.

[0332] As discussed above, a hairpin containing molecule having adouble-stranded region may be used as RNAi. The length of the doublestranded region may be from about 20 bp to about 2.5 kbp in length, fromabout 20 bp to about 2 kbp in length, 20 bp to about 1.5 kbp in length,from about 20 bp to about 1 kbp in length, 20 bp to about 750 bp inlength, from about 20 bp to about 500 bp in length, 20 bp to about 400bp in length, 20 bp to about 300 bp in length, 20 bp to about 250 bp inlength, from about 20 bp to about 200 bp in length, from about 20 bp toabout 150 bp in length, from about 20 bp to about 100 bp in length, 20bp to about 90 bp in length, 20 bp to about 80 bp in length, 20 bp toabout 70 bp in length, 20 bp to about 60 bp in length, 20 bp to about 50bp in length, 20 bp to about 40 bp in length, 20 bp to about 30 bp inlength, from about 20 bp to about 25 bp in length, from about 15 bp toabout 25 bp in length, from about 17 bp to about 25 bp in length, fromabout 19 bp to about 25 bp in length, from about 15 bp to about 23 bp,from about 17 bp to about 23 bp, from about 19 bp to about 23 bp inlength, from about 15 bp to about 21 bp, from about 17 bp to about 21bp, or from about 19 bp to about 21 bp in length. The non-base-pairedportion of the hairpin (i.e., loop) can be of any length that permitsthe two regions of homology that make up the double-stranded portion ofthe hairpin to fold back upon one another.

[0333] Any suitable promoter may be used to control the production ofRNA from the nucleic acid molecules of the invention. Promoters may bethose recognized by any polymerase enzyme. For example, promoters may bepromoters for RNA polymerase II or RNA polymerase III (e.g., a U6promoter, an H1 promoter, etc.). Other suitable promoters include, butare not limited to, T7 promoter, cytomegalovirus (CMV) promoter, mousemammary tumor virus (MMTV) promoter, metalothionine, RSV (Rous sarcomavirus) long terminal repeat, SV40 promoter, human growth hormone (hGH)promoter. Other suitable promoters are known to those skilled in the artand are within the scope of the present invention.

[0334] One example of a construct designed to produce RNAi is shown inFIG. 5B. In this construct, a DNA segment is inserted into a vector suchthat RNA corresponding to both strands are produced as two separatetranscripts. Another example of a construct designed to produce RNAi isshown in FIG. 5C. In this construct, two copies of a DNA segment areinserted into a vector such that RNA corresponding to both strands areagain produced. Yet another example of a construct designed to produceRNAi is shown in FIG. 5D. In this construct, two copies of a DNA segmentare inserted into a vector such that RNA corresponding to both strandsare produced as a single transcript. The exemplary vector system shownin shown in FIGS. 5E and 5F comprises two vectors, each of which containcopies of the same DNA segment. Expression of one of these DNA segmentsresults in the production of sense RNA while expression of the otherresults in the production of an anti-sense RNA. RNA strands producedfrom vectors represented in FIGS. 5B-5F will thus have complementarynucleotide sequences and will generally hybridize either to each orintramolecularly under physiological conditions.

[0335] Nucleic acid segments designed to produce RNAi, such as thevectors represented in FIGS. 5B-5F, need not correspond to thefull-length gene or open reading frame. For example, when the nucleicacid segment corresponds to an ORF, the segment may only correspond topart of the ORF (e.g., 50 nucleotides at the 5′ or 3′ end of the ORF).Further, while FIGS. 5B-5F show vectors designed to produce RNAi,nucleic acid segments may also perform the same function in other forms(e.g., when inserted into the chromosome of a host cell).

[0336] Gene silencing methods involving the use of compounds such asRNAi and antisense RNA, for examples, are particularly useful foridentifying gene functions. More specifically, gene silencing methodscan be used to reduce or prevent the expression of one or more genes ina cell or organism. Phenotypic manifestations associated with theselective inhibition of gene functions can then be used to assign roleto the “silenced” gene or genes. As an example, Chuang, et al., Proc.Natl. Acad. Sci. (USA) 97:4985-4990 (2000), have demonstrated that invivo production of RNAi can alter gene activity in Arabidopsis thaliana.Thus, the invention provides methods for regulating expression ofnucleic acid molecules in cells and tissues comprising the expression ofRNAi and antisense RNA. The invention further provides methods forpreparing nucleic acid molecules which can be used to produce RNAcorresponding to one or both strands of a DNA molecule.

[0337] Further, viral vectors and/or nucleic acids molecules of theinvention may be used to insert into cells nucleic acid segments thatencode expression products involved in each step of particularbiological pathways (e.g., biosynthesis of amino acids such as lysine,threonine, etc.) or expression products involved in one or a few stepsof such pathways. These nucleic acid molecules can be designed to, ineffect, amplify genes encoding expression products in such pathways,insert genes into cells that encode expression products involved inpathways not normally found in the cells, or to replace one or moregenes involved one or more steps of particular biological pathways incells. Thus, gene therapy vectors of the invention may contain nucleicacid that results in the production one or more products (e.g., one,two, three, four, five, eight, ten, fifteen, etc.). Such vectors,especially those that lead to the production of more than one product,will be particularly useful for the treatment of diseases and/orconditions that result from the expression and/or lack of expression ofmore than one gene or for the treatment of more than one diseases and/orconditions.

[0338] Thus, in related aspects, the invention provides gene therapyvectors that express one or more expression products (e.g., one or morefusion proteins), methods for producing such vectors, methods forperforming gene therapy using vectors of the invention, expressionproducts of such vector (e.g., encoded RNA and/or proteins), and hostcells that contain vectors of the invention.

[0339] For general reviews of the methods of gene therapy, seeGoldspiel, et al., Clinical Pharmacy 12:488-505 (1993); Wu and Wu,Biotherapy 3:87-95 (1991); Tolstoshev, Ann. Rev. Pharmacol. Toxicol.32:573-596 (1993); Mulligan, Science 260:926-932 (1993); and Morgan andAnderson, Ann. Rev. Biochem. 62:191-217 (1993); May, TIBTECH11(5):155-215 (1993)). Methods commonly known in the art of recombinantDNA technology that can be used are described in Ausubel, et al. (eds.),1993, Current Protocols in Molecular Biology, John Wiley & Sons, NY; andKriegler, 1990, Gene Transfer and Expression, A Laboratory Manual,Stockton Press, N.Y.

[0340] Delivery of the viral vectors and/or nucleic acids molecules ofthe invention into a patient may be either direct, in which case thepatient is directly exposed to the nucleic acids and/or viral vectors ofthe invention, or indirect, in which case, cells are firsttransfected/transduced with the nucleic acid/viral vector in vitro, thentransplanted into the patient. These two approaches are known,respectively, as in vivo or ex vivo gene therapy.

[0341] In another specific embodiment, viral vectors that containnucleic acid sequences encoding an antibody or other antigen-bindingprotein of the invention are used. The nucleic acid sequences encodingthe antibody to be used in gene therapy are cloned into one or moreviral vectors, which facilitates delivery of the gene into a patient.

[0342] Adenoviruses are examples of viruses that can be used to prepareviral vectors that can be used in gene therapy. Adenoviral vectors areespecially attractive vehicles for delivering genes to respiratoryepithelia and the use of such vectors are included within the scope ofthe invention. Adenoviruses naturally infect respiratory epithelia wherethey cause a mild disease. Other targets for adenovirus-based deliverysystems are liver, the central nervous system, endothelial cells, andmuscle. Adenoviral vectors have the advantage of being capable ofinfecting non-dividing cells. Kozarsky and Wilson, Current Opinion inGenetics and Development 3:499-503 (1993) present a review ofadenovirus-based gene therapy. Bout, et al., Human Gene Therapy 5:3-10(1994) demonstrated the use of adenoviral vectors to transfer genes tothe respiratory epithelia of rhesus monkeys. Other instances of the useof adenoviral vectors in gene therapy can be found in Rosenfeld, et al.,Science 252:431-434 (1991); Rosenfeld, et al., Cell 68:143-155 (1992);Mastrangeli, et al., J. Clin. Invest. 91:225-234 (1993); PCT PublicationNos. WO 94/12649 and WO 96/17053; U.S. Pat. No. 5,998,205; and Wang, etal., Gene Therapy 2:775-783 (1995), the disclosures of all of which areincorporated herein by reference in their entireties. In a oneembodiment, adenoviral vectors are used for in vivo gene therapy.

[0343] Another approach to gene therapy involves transferring a gene tocells in tissue culture, for example, by infection with a viral vectorof the present invention. The viral vector may contain a sequenceencoding a therapeutic polypeptide or nucleic acid (i.e., antisensemolecule) and may further include a sequence encoding a selectablemarker. The cells are then placed under selection to isolate those cellsthat have taken up and are expressing the transferred gene. Those cellsare then delivered to a patient.

[0344] In this embodiment, the viral vector is introduced into a cellprior to administration in vivo of the resulting recombinant cell. Theresulting recombinant cells can be delivered to a patient by variousmethods known in the art. Recombinant blood cells (e.g., hematopoieticstem or progenitor cells) will generally be administered intravenously.The amount of cells envisioned for use depends on the desired effect,patient state, etc., and can be determined by one skilled in the art.

[0345] Cells into which a viral vector can be introduced for purposes ofgene therapy encompass any desired, available cell type, and include butare not limited to epithelial cells, endothelial cells, keratinocytes,fibroblasts, muscle cells, hepatocytes; blood cells such asT-lymphocytes, B-lymphocytes, monocytes, macrophages, neutrophils,eosinophils, megakaryocytes, granulocytes; various stem or progenitorcells, in particular hematopoietic stem or progenitor cells (e.g., asobtained from bone marrow, umbilical cord blood, peripheral blood, fetalliver, etc.).

[0346] In a certain embodiment, the cell used for gene therapy isautologous to the patient.

[0347] In an embodiment in which recombinant cells are used in genetherapy, viral vectors containing nucleic acids encoding an antibody orother antigen-binding protein are introduced into the cells such thatthey are expressible by the cells and/or their progeny, and therecombinant cells are then administered in vivo for therapeutic effect.In a specific embodiment, stem or progenitor cells are used. Any stemand/or progenitor cells that can be isolated and maintained in vitro canpotentially be used in accordance with this embodiment of the presentinvention (see, e.g., PCT Publication WO 94/08598, dated Apr. 28, 1994;Stemple and Anderson, Cell 71:973-985 (1992); Rheinwald, Meth. Cell Bio.21A:229 (1980); and Pittelkow and Scott, Mayo Clinic Proc. 61:771(1986)).

[0348] In a specific embodiment, viral vectors and/or nucleic acidsmolecules of the invention comprise nucleic acid sequences to beintroduced for purposes of gene therapy under the control of aninducible promoter operably linked to the coding region, such thatexpression of the nucleic acid sequences is controllable by controllingthe presence or absence of the appropriate inducer of transcription.

[0349] The viral vectors and/or nucleic acids molecules of the inventioncan also be used to produce transgenic organisms (e.g., animals).Animals of any species, including, but not limited to, mice, rats,rabbits, hamsters, guinea pigs, pigs, micro-pigs, goats, sheep, cows andnon-human primates (e.g., baboons, monkeys, and chimpanzees) may be usedto generate transgenic animals. Viruses capable of infecting the desiredcell type are known to those skilled in the art and viral vectors basedon these viruses may be used in the methods of the invention.

[0350] The present invention provides for transgenic organisms thatcarry the viral vectors and/or nucleic acids molecules of the inventionor nucleic acid sequences provided by the viral vectors and/or nucleicacids molecules of the invention in all their cells, as well asorganisms that carry these viral vectors or sequences in some, but notall, of their cells, i.e., mosaic organisms or chimeric. The viralvectors and/or nucleic acids molecules of the invention may beintegrated as a single copy or as multiple copies. The viral vectorsand/or nucleic acids molecules of the invention may also be selectivelyintroduced into and activated in a particular cell type by following,for example, the teaching of Lasko, et al. (Lasko, et al., Proc. Natl.Acad. Sci. USA 89:6232-6236 (1992)). The regulatory sequences requiredfor such a cell-type specific activation will depend upon the particularcell type of interest, and will be apparent to those of skill in theart. When it is desired that the sequences of interest contained in theviral vectors and/or nucleic acids molecules of the invention beintegrated into the chromosomal site of the endogenous gene, this willnormally be done by gene targeting. Briefly, when such a technique is tobe utilized, viral vectors containing some nucleotide sequenceshomologous to the endogenous gene are designed for the purpose ofintegrating, via homologous recombination with chromosomal sequences,into and disrupting the function of the nucleotide sequence of theendogenous gene. Viral vectors and/or nucleic acids molecules of theinvention may also be selectively introduced into a particular celltype, thus inactivating the endogenous gene in only that cell type, byfollowing, for example, the teaching of Gu, et al. (Gu, et al., Science265:103-106 (1994)). The regulatory sequences required for such acell-type specific inactivation will depend upon the particular celltype of interest, and will be apparent to those of skill in the art. Thecontents of each of the documents recited in this paragraph is hereinincorporated by reference in its entirety.

[0351] Once transgenic organisms have been generated, the expression ofthe recombinant gene may be assayed utilizing standard techniques.Initial screening may be accomplished by Southern blot analysis or PCRtechniques to analyze organism tissues to verify that integration ofnucleic acid molecules of the invention has taken place. The level ofmRNA expression of nucleic acid sequences introduced by the viralvectors and/or nucleic acids molecules of the invention in the tissuesof the transgenic organisms may also be assessed using techniquesincluding, but not limited to, Northern blot analysis of tissue samplesobtained from the organism, in situ hybridization analysis, and reversetranscriptase-PCR (RT-PCR). Samples of tissue that express the insertedsequences may also be evaluated immunocytochemically orimmunohistochemically using antibodies specific for the expressionproduct of these nucleic acid molecules.

[0352] Once the founder organisms are produced, they may be bred,inbred, outbred, or crossbred to produce colonies of the particularorganism. Examples of such breeding strategies include, but are notlimited to: outbreeding of founder organisms with more than oneintegration site in order to establish separate lines; inbreeding ofseparate lines in order to produce compound transgenic organisms thatexpress sequences of interest at higher levels because of the effects ofadditive expression of each copy of nucleic acid molecules of theinvention; crossing of heterozygous transgenic organisms to produceorganisms homozygous for a given integration site in order to bothaugment expression and eliminate the need for screening of organisms byDNA analysis; crossing of separate homozygous lines to produce compoundheterozygous or homozygous lines; and breeding to place the nucleic acidmolecules of the invention on a distinct background that is appropriatefor an experimental model of interest.

[0353] Transgenic and “knock-out” organisms of the invention have usesthat include, but are not limited to, model systems (e.g., animal modelsystems) useful in elaborating the biological function of expressionproducts of sequences of interest, studying conditions and/or disordersassociated with aberrant expression of expression products of sequencesof interest, and in screening for compounds effective in amelioratingsuch conditions and/or disorders.

[0354] As one skilled in the art would recognize, in many instances whenviral vectors containing sequences of interest are introduced intometazoan organisms, it will be desirable to operably link the sequencesthat encode expression products to tissue-specific transcriptionalregulatory sequences (e.g., tissue-specific promoters) where productionof the expression product is desired. Such promoters can be used tofacilitate production of these expression products in desired tissues. Aconsiderable number of tissue-specific promoters are known in the art.

[0355] Host Cells

[0356] The invention also relates to host cells comprising one or moreof the viral vectors and/or nucleic acids molecules of the inventioncontaining one or more sequences of interest (e.g., two, three, four,five, seven, ten, twelve, fifteen, twenty, thirty, fifty, etc.),particularly those viral vectors described in detail herein.Representative host cells that may be used according to this aspect ofthe invention include, but are not limited to, bacterial cells, yeastcells, plant cells and animal cells. Preferred bacterial host cellsinclude Escherichia spp. cells (particularly E. coli cells and mostparticularly E. coli strains DH10B, Stbl2, DH5α, DB3, DB3.1 (preferablyE. coli LIBRARY EFFICIENCYS® DB3.1™ Competent Cells; InvitrogenCorporation, Carlsbad, Calif.), DB4, DB5, JDP682 and ccdA-over (see U.S.application Ser. No. 09/518,188, filed Mar. 2, 2000, and U.S.provisional Application No. 60/475,004, filed Jun. 3, 2003, by LouisLeong et al., entitled “Cells Resistant to Toxic Genes and UsesThereof,” the disclosures of which are incorporated by reference hereinin their entireties); a DB3 cell (deposit number NRRL B-30097), a DB3.1cell (deposit number NRRL B-30098), a DB4 cell (deposit number NRRLB-30106), a DB5 cell (deposit number NRRL B-30107), a JDP682 cell(deposit number NRRL B-30667), a ccdA-over cell (deposit number NRRLB-30668), or a mutant or derivative thereof; Bacillus spp. cells(particularly B. subtilis and B. megaterium cells), Streptomyces spp.cells, Erwinia spp. cells, Klebsiella spp. cells, Serratia spp. cells(particularly S. marcessans cells), Pseudomonas spp. cells (particularlyP. aeruginosa cells), and Salmonella spp. cells (particularly S.typhimurium and S. typhi cells). Preferred animal host cells includeinsect cells (most particularly Drosophila melanogaster cells,Spodoptera frugiperda Sf9 and Sf21 cells and Trichoplusa High-Fivecells), nematode cells (particularly C. elegans cells), avian cells,amphibian cells (particularly Xenopus laevis cells), reptilian cells,and mammalian cells (most particularly NIH3T3, 293, CHO, COS, VERO, BHKand human cells). Preferred yeast host cells include Saccharomycescerevisiae cells and Pichia pastoris cells. These and other suitablehost cells are available commercially, for example, from InvitrogenCorporation, (Carlsbad, Calif.), American Type Culture Collection(Manassas, Va.), and Agricultural Research Culture Collection (NRRL;Peoria, Ill.).

[0357] Nucleic acid molecules to be used in the present invention maycomprise one or more origins of replication (ORIs), and/or one or moreselectable markers. In some embodiments, molecules may comprise two ormore ORIs at least two of which are capable of functioning in differentorganisms (e.g., one in prokaryotes and one in eukaryotes). For example,a nucleic acid may have an ORI that functions in one or more prokaryotes(e.g., E. coli, Bacillus, etc.) and another that functions in one ormore eukaryotes (e.g., yeast, insect, mammalian cells, etc.). Selectablemarkers may likewise be included in nucleic acid molecules of theinvention to allow selection in different organisms. For example, anucleic acid molecule may comprise multiple selectable markers, one ormore of which functions in prokaryotes and one or more of whichfunctions in eukaryotes.

[0358] Methods for introducing the viral vectors and/or nucleic acidsmolecules of the invention into the host cells described herein, toproduce host cells comprising one or more of the viral vectors and/ornucleic acids molecules of the invention, will be familiar to those ofordinary skill in the art. For instance, the nucleic acid moleculesand/or viral vectors of the invention may be introduced into host cellsusing well known techniques of infection, transduction, electroporation,transfection, and transformation. The nucleic acid molecules and/orviral vectors of the invention may be introduced alone or in conjunctionwith other nucleic acid molecules and/or vectors and/or proteins,peptides or RNAs. Alternatively, the nucleic acid molecules and/or viralvectors of the invention may be introduced into host cells as aprecipitate, such as a calcium phosphate precipitate, or in a complexwith a lipid. Electroporation also may be used to introduce the nucleicacid molecules and/or viral vectors of the invention into a host.Likewise, such molecules may be introduced into chemically competentcells such as E. coli. If the vector is a virus, it may be packaged invitro or introduced into a packaging cell and the packaged virus may betransduced into cells. Thus nucleic acid molecules of the invention maycontain and/or encode one or more packaging signal (e.g., viralpackaging signals that direct the packaging of viral nucleic acidmolecules). Hence, a wide variety of techniques suitable for introducingthe nucleic acid molecules and/or vectors of the invention into cells inaccordance with this aspect of the invention are well known and routineto those of skill in the art. Such techniques are reviewed at length,for example, in Sambrook, J., et al., Molecular Cloning, a LaboratoryManual, 2nd Ed., Cold Spring Harbor, N.Y.: Cold Spring Harbor LaboratoryPress, pp. 16.30-16.55 (1989), Watson, J. D., et al., Recombinant DNA,2nd Ed., New York: W. H. Freeman and Co., pp. 213-234 (1992), andWinnacker, E.-L., From Genes to Clones, New York: VCH Publishers (1987),which are illustrative of the many laboratory manuals that detail thesetechniques and which are incorporated by reference herein in theirentireties for their relevant disclosures.

[0359] Kits

[0360] In another aspect, the invention provides kits that may be usedin conjunction with methods the invention. Kits according to this aspectof the invention may comprise one or more containers, which may containone or more components selected from the group consisting of one or morenucleic acid molecules (e.g., one or more nucleic acid moleculescomprising one or more viral sequences and /or one or more recombinationsites) and/or viral vectors of the invention, one or more primers, themolecules and/or compounds of the invention, one or more polymerases,one or more reverse transcriptases, one or more recombination proteins(or other enzymes for carrying out the methods of the invention), one ormore ligases, one or more buffers, one or more detergents, one or morerestriction endonucleases, one or more nucleotides, one or moreterminating agents (e.g., ddNTPs), one or more transfection reagents,pyrophosphatase, and the like.

[0361] A wide variety of nucleic acid molecules and/or viral vectors ofthe invention can be used with the invention. Further, due to themodularity of the invention, these nucleic acid molecules can becombined in wide range of ways. Examples of nucleic acid molecules thatcan be supplied in kits of the invention include those that containpromoters, signal peptides, enhancers, repressors, selection markers,transcription signals, translation signals, primer hybridization sites(e.g., for sequencing or PCR), recombination sites, restriction sitesand polylinkers, sites that suppress the termination of translation inthe presence of a suppressor tRNA, suppressor tRNA coding sequences,sequences that encode domains and/or regions (e.g., 6 His tag) for thepreparation of fusion proteins, origins of replication, telomeres,centromeres, and the like. Similarly, libraries can be supplied in kitsof the invention. These libraries may be in the form of replicablenucleic acid molecules or they may comprise nucleic acid molecules thatare not associated with an origin of replication. As one skilled in theart would recognize, the nucleic acid molecules of libraries, as well asother nucleic acid molecules that are not associated with an origin ofreplication, either could be inserted into other nucleic acid moleculesthat have an origin of replication or would be an expendable kitcomponents.

[0362] Further, in some embodiments, libraries supplied in kits of theinvention may comprise two components: (1) the nucleic acid molecules ofthese libraries and (2) 5′ and/or 3′ recombination sites. In someembodiments, when the nucleic acid molecules of a library are suppliedwith 5′ and/or 3′ recombination sites, it will be possible to insertthese molecules into nucleic acid molecules comprising all or a portionof a viral genome, which also may be supplied as a kit component, usingrecombination reactions. In other embodiments, recombination sites canbe attached to the nucleic acid molecules of the libraries before use(e.g., by the use of a ligase, which may also be supplied with the kit).In such cases, nucleic acid molecules that contain recombination sitesor primers that can be used to generate recombination sites may besupplied with the kits.

[0363] Nucleic acid molecules comprising all or a portion of a viralgenome to be supplied in kits of the invention can vary greatly. In someinstances, these molecules will contain an origin of replication, atleast one selectable marker, and at least one recombination site. Forexample, molecules supplied in kits of the invention can have fourseparate recombination sites that allow for insertion of sequence ofinterest at two different locations of a nucleic acid molecule, forexample, as shown in FIG. 2. Other attributes of vectors supplied inkits of the invention are described elsewhere herein.

[0364] In some embodiments, the kits of the invention may comprise aplurality of containers, each container comprising one or more nucleicacid segments comprising viral sequences and/or one or morerecombination sites and/or topoisomerase recognition sites. Segments maybe provided with recombination sites such that a series of segments(e.g., two, three, four, five six, seven, eight, nine, ten, etc.) may becombined in order to construct a viral vector or other nuclei acidmolecule of the present invention. Segments may be combined in reactionsinvolving two or more segments (e.g., three, four, five, six, seven,eight, nine, ten, etc.). Each individual segment may be, independentlyof any other segment, from about 100 bp to about 35 kb in length, orfrom about 100 bp to about 20 kb in length, or from about 100 bp toabout 10 kb in length, or from about 100 bp to about 5 kb in length, orfrom about 100 bp to about 2.5 kb in length, or from about 100 bp toabout 1 kb in length, or from about 100 bp to about 500 bp in length.The present invention also contemplates methods for assembling and usingsuch segments, nucleic acid molecules assembled by such methods, andcompositions comprising such nucleic acid molecules.

[0365] Segments may be prepared so as to contain viral transcriptionunits. For example, when an adenoviral vector is to be prepared, onesegment may comprise, in addition to one or more recombination sitesand/or one or more topoisomerase recognition sites, sequencescorresponding to the E1 region, the E2 region, the E3 region, and/or theE4 region. Other segments may comprise sequences corresponding to one ormore late transcription units and/or viral inverted terminal repeats.Segments comprising nucleic acid sequences of interest may be preparedso as to construct a viral vector or other nucleic acid molecule inwhich one or more viral nucleic acid sequences, present in a wild-typevirus, are not present in the viral vector. Segments comprising anucleic acid sequence of interest may be prepared and inserted into aviral vector in place of one or more segments comprising viralsequences. In some embodiments, sequences that are present in awild-type virus but not present in the viral vectors of the inventionare those that are not required for replication in cultured cells. Forexample, a segment comprising a nucleic acid sequence of interest may beused to construct an adenoviral vector wherein the nucleic acid sequenceof interest replaces one or more of the E1 region and/or the E3 region.Where necessary (e.g., in the case of the E1 functions) viral functionsrequired to support replication of the viral vector may be supplied intrans (e.g., from the genome of the host cell). Segments may be preparedto construct viral vectors wherein a nucleic acid sequence of interestis place in the viral genome in a position known to be tolerant ofnucleic acid insertions, for example, upstream of the E4 region.

[0366] A kit of the present invention may comprise a containercontaining a nucleic acid molecule comprising all or a portion of aviral genome and comprising two recombination sites that do notrecombine with each other. The recombination sites may flank aselectable marker that allows selection for or against the presence ofthe nucleic acid molecule in a host cell or identification of a hostcell containing or not containing the nucleic acid. A nucleic acidmolecule to be included in a kit may comprise more than tworecombination sites, for example, a nucleic acid molecule may comprisemultiple pairs of recombination sites (e.g., two, three, four, five,six, seven, eight, nine, ten, etc.) where members of a pair ofrecombination sites do not recombine or substantially recombine witheach other. In some embodiments, members of one pair of recombinationsites do not recombine with members of another pair present in the samenucleic acid molecule.

[0367] Kits of the invention may comprise containers containing one ormore recombination proteins. Suitable recombination proteins have beendisclosed above and include, but are not limited to, Cre, Int, IHF, Xis,Flp, Fis, Hin, Gin, Cin, Tn3 resolvase, ΦC31, TndX, XerC, and XerD.

[0368] Kits of the invention may also comprise one or more topoisomeraseproteins and/or one or more nucleic acids comprising one or moretopoisomerase recognition sequence. Suitable topoisomerases include TypeIA topoisomerases, Type IB topoisomerases and/or Type II topoisomerases.Suitable topoisomerases include, but are not limited to, poxvirustopoisomerases, including vaccinia virus DNA topoisomerase I, E. colitopoisomerase III, E. coli topoisomerase I, topoisomerase HII,eukaryotic topoisomerase II, archeal reverse gyrase, yeast topoisomeraseIII, Drosophila topoisomerase III, human topoisomerase III,Streptococcus pneumoniae topoisomerase III, bacterial gyrase, bacterialDNA topoisomerase IV, eukaryotic DNA topoisomerase II, and T-even phageencoded DNA topoisomerases, and the like. Suitable recognition sequenceshave been described above.

[0369] In use, a nucleic acid molecule comprising all or a portion of aviral genome provided in a kit of the invention may be combined with anucleic acid molecule comprising a sequence of interest usingrecombinational cloning. The nucleic acid molecule comprising all or aportion of a viral genome may be provided, for example, with tworecombination sites that do not recombine with each other. The nucleicacid molecule comprising a sequence of interest may also be providedwith two recombination sites, each of which is capable of recombiningwith one of the two sites present on the a nucleic acid moleculecomprising all or a portion of a viral genome. In the presence of theappropriate recombination proteins, the nucleic acid molecule reactswith the nucleic acid molecule comprising all or a portion of a viralgenome in order to form a recombinant nucleic acid molecule containingthe sequence of interest and all or a portion of a viral genome. Whenthe nucleic acid molecule comprising all or a portion of a viral genomecomprises multiple pairs of recombination sites, multiple nucleic acidmolecules comprising sequences of interest, which may be the same ordifferent, may be combined with the nucleic acid molecule comprising allor a portion of a viral genome in order to form a nucleic acid moleculecomprising all or a portion of a viral genome and also comprisesmultiple sequence of interest.

[0370] Kits of the invention can also be supplied with primers. Theseprimers will generally be designed to anneal to molecules havingspecific nucleotide sequences. For example, these primers can bedesigned for use in PCR to amplify a particular nucleic acid molecule.Further, primers supplied with kits of the invention can be sequencingprimers designed to hybridize to vector sequences. Thus, such primerswill generally be supplied as part of a kit for sequencing nucleic acidmolecules that have been inserted into a vector.

[0371] One or more buffers (e.g., one, two, three, four, five, eight,ten, fifteen) may be supplied in kits of the invention. These buffersmay be supplied at a working concentrations or may be supplied inconcentrated form and then diluted to the working concentrations. Thesebuffers will often contain salt, metal ions, co-factors, metal ionchelating agents, etc. for the enhancement of activities of thestabilization of either the buffer itself or molecules in the buffer.Further, these buffers may be supplied in dried or aqueous forms. Whenbuffers are supplied in a dried form, they will generally be dissolvedin water prior to use.

[0372] Kits of the invention may contain virtually any combination ofthe components set out above or described elsewhere herein. As oneskilled in the art would recognize, the components supplied with kits ofthe invention will vary with the intended use for the kits. Thus, kitsmay be designed to perform various functions set out in this applicationand the components of such kits will vary accordingly.

[0373] Kits of the invention may comprise one or more pages of writteninstructions for carrying out the methods of the invention. For example,instructions may comprise methods steps necessary to carry outrecombinational cloning of an ORF provided with recombination sites anda vector also comprising recombination sites and optionally furthercomprising one or more functional sequences.

[0374] It will be understood by one of ordinary skill in the relevantarts that other suitable modifications and adaptations to the methodsand applications described herein are readily apparent from thedescription of the invention contained herein in view of informationknown to the ordinarily skilled artisan, and may be made withoutdeparting from the scope of the invention or any embodiment thereof.Having now described the present invention in detail, the same will bemore clearly understood by reference to the following examples, whichare included herewith for purposes of illustration only and are notintended to be limiting of the invention.

[0375] The entire disclosures of U.S. appl. Ser. No. 08/486,139, (nowabandoned), filed Jun. 7, 1995, U.S. appl. Ser. No. 08/663,002, filedJun. 7, 1996 (now U.S. Pat. No. 5,888,732), U.S. appl. Ser. No.09/233,492, filed Jan. 20, 1999, (now U.S. Pat. No. 6,270,969), U.S.appl. Ser. No. 09/233,493, filed Jan. 20, 1999, (now U.S. Pat. No.6,143,557), U.S. appl. Ser. No. 09/005,476, filed Jan. 12, 1998, (nowU.S. Pat. No. 6,171,861), U.S. appl. Ser. No. 09/432,085 filed Nov. 2,1999, U.S. appl. Ser. No. 09/498,074 filed Feb. 4, 2000, U.S. Appl. No.60/065,930, filed Oct. 24, 1997, U.S. appl. Ser. No. 09/177,387, filedOct. 23, 1998, U.S. appl. Ser. No. 09/296,280, filed Apr. 22, 1999, (nowU.S. Pat. No. 6,277,608), U.S. appl. Ser. No. 09/296,281, filed Apr. 22,1999, (now abandoned), U.S. appl. Ser. No. 09/648,790, filed Aug. 28,2000, U.S. appl. Ser. No. 09/732,914 (published as US 2002 0007051),filed Dec. 11, 2000, U.S. appl. Ser. No. 09/855,797, filed May 16, 2001,U.S. appl. Ser. No. 09/907,719, filed Jul. 19, 2001, U.S. appl. Ser. No.09/907,900, filed Jul. 19, 2001, U.S. appl. Ser. No. 09/985,448, filedNov. 2, 2001, U.S. Appl. No. 60/108,324, filed Nov. 13, 1998, U.S. appl.Ser. No. 09/438,358, filed Nov. 12, 1999, U.S. Appl. No. 60/161,403,filed Oct. 25, 1999, U.S. appl. Ser. No. 09/695,065, filed Oct. 25,2000, U.S. appl. Ser. No. 09/984,239, filed Oct. 29, 2001, U.S. Appl.No. 60/122,389, filed Mar. 2, 1999, U.S. Appl. Ser. No. 60/126,049,filed Mar. 23, 1999, U.S. Appl. No. 60/136,744, filed May 28, 1999, U.S.appl. Ser. No. 09/517,466, filed Mar. 2, 2000, U.S. Appl. No.60/122,392, filed Mar. 2, 1999, U.S. appl. Ser. No. 09/518,188, filedMar. 2, 2000, U.S. Appl. No. 60/169,983, filed Dec. 10, 1999, U.S. Appl.No. 60/188,000, filed Mar. 9, 2000, U.S. appl. Ser. No. 09/732,914,filed Dec. 11, 2001, U.S. Appl. No. 60/284,528, filed Apr. 19, 2001,U.S. Appl. No. 60/291,973, filed May 21, 2001, U.S. Appl. No.60/318,902, filed Sep. 14, 2001, U.S. Appl. No. 60/333,124, filed Nov.27, 2001, and U.S. appl. Ser. No. 10/005,876, filed Dec. 7, 2001, areherein incorporated by reference.

EXAMPLES

[0376] The present invention provides an extremely versatile method forthe modular construction of nucleic acids and production ofpolypeptides. Both insert nucleic acid segments and the vector cancontain sequences selected so as to confer desired characteristics onthe product molecules. In some embodiments, in addition to the insert,one or more of the portions of the nucleic acid comprising all or aportion of a viral genome adjacent to the insert, can contain one ormore selected sequences. The selected sequences might encode ribozymes,epitope tags, structural domains, selectable markers, internal ribosomeentry sequences, promoters, enhancers, recombination sites and the like.

[0377] In some embodiments, more than one sequence of interest may beincorporated in a nucleic acid molecule comprising all or a portion of aviral genome. The incorporated sequences of interest may be adjacent toone another or may be separated by a portion of the nucleic acidmolecule comprising all or a portion of a viral genome. When separated,the portion of the nucleic acid molecule separating the sequences ofinterest may comprise one or more selectable markers flanked by areactive pair of recombination sites in addition to containing therecombination sites used to insert the nucleic acid segments. Theportion of the nucleic acid molecule separating the sequences ofinterest may also comprise viral sequences and/or other sequencesconferring a desired characteristic on the nucleic acid molecule and/orsequences of interest.

[0378] A sequence of interest may be a sequence of any type. Forexample, the sequence may encode one or more polypeptides and/or maycontain one or more un-translated regions. Sequences of interest may betranscribed and translated into polypeptides or may be transcribed andnot translated into polypeptides, for example, anti-sense molecules,ribozymes, and RNAi. Sequences of interest may or may not comprise astop codon. Sequences comprising a stop codon may or may not compriseadditional sequences 3′ to the stop codon that may be in frame withsequences 5′ to the stop codon. In some embodiments, stop codons may besuppressed in order to produce a fusion polypeptide.

[0379] Throughout this disclosure, the term gene of interest (GOI) maybe used for the sake of convenience. This should not be construed aslimiting the present invention to nucleic acid sequences comprisinggenes. Any nucleic acid sequence of interest can be inserted into avector of the invention using materials and methods described herein.

EXAMPLE 1 Preparation of a Viral Vector of the Invention

[0380]FIG. 6 is a plasmid map of the pAd/CMV/V5-DEST vector, one exampleof a nucleic acid comprising all or a portion of a viral genomeaccording to the present invention. The nucleotide sequence of theplasmid is provided in Table 6 (SEQ ID NO:). The plasmid contains thefirst 458 nucleotides of Ad5, including the left ITR and packagingsequence, followed the cytomegalovirus promoter (CMV) and the T7promoter. The promoters are followed by a sequence containing selectablemarkers flanked by recombination sites attR1 and attR2. Any othersuitable pair of recombination sites might be employed as long as theyare selected so as not to recombine with each other. After the attR2site, the V5 epitope coding sequence is followed by stop codons in allthree reading frames and the herpes virus thymidine kinasepolyadenylation signal. This is followed by the nucleotides fromposition 3513 to the right end of the adenoviral genome including theright ITR. After the adenoviral sequences, are plasmid sequencesincluding a plasmid origin of replication followed by the ampicillinresistance gene. The plasmid sequences are flanked by Pacd restrictionenzyme recognition sites. Thus, after replacement of the replaceablesequence with a sequence of interest flanked by attL1 and attL2 in arecombination reaction, an infectious viral genome can be prepared bydigestion of the recombination reaction product with Pacl to remove theplasmid sequences. In this particular embodiment, the viral genome is anadenoviral genome deleted in the E1 and E3 regions. The E1 function mustbe supplied in trans in order for the virus to replicate, for example,from the host cell as in 293 cells. The gene products of the E3 regionare not required for replication.

[0381] In order to prepare a viral vector according to the presentinvention, a particular sequence of interest may be prepared withrecombination sites compatible to those in the pAd/CMV/V5-DEST vector.This may be accomplished using standard techniques, for example, byamplifying a sequences of interest with primers comprising theappropriate recombination site sequences. If a PCR product contains theappropriate recombination site sequences, it may be used directly in arecombination reaction. Optionally, a PCR product or other nucleic acidcomprising the sequence of interest may be cloned into a GATEWAY™ entryvector. This can be accomplished using any conventional technique, forexample, by a) traditional restriction fragment ligation, b)TOPO-mediated cloning of the nucleic acid comprising the sequence ofinterest into pENTR-dTOPO, or c) GATEWAY™ clonase reaction betweenPCR-amplified sequence of interest (e.g., gene of interest (GOI))containing flanking attB sites with pDONR DNA. Any of these threemethods will result in the sequence of interest being inserted into anentry vector. Using the terminology of the GATEWAY™ Technology, theresultant vector would be designated pENTR-GOI for an entry vectorcomprising a gene of interest (GOI). This should not be construed aslimiting the sequences of interest to those encoding genes; any sequenceof interest may be inserted into a pENTR vector in this fashion. In thisexample, this results in the sequence of interest being flanked by attL1and attL2 recombination sites.

[0382] In an in vitro GATEWAY™ LR reaction, the pENTR-GOI vector may becombined with pAd-CMV-DEST. The reaction may be incubated for anappropriate period of time, for example, 1 hour at room temperature.This reaction moves the sequence of interest into the adenoviral vector,pAd-CMV-DEST.

[0383] The adenoviral vector containing a sequence of interest is usedto transform competent bacteria (i.e., DH5α, TOP10, HB101, etc.). All ora portion of the LR reaction mixture is used to transform competentbacteria and the transformed bacteria are plated on LB-ampicillinbacterial plates and incubated overnight at 37° C.

[0384] Several bacterial colonies—2-4 is usually sufficient—may bepicked and used to inoculate overnight cultures in LB-ampicillin liquidmedium and grown overnight at 37° C.

[0385] Plasmid DNA is prepared from the cultures using conventionaltechniques and analyzed for the presence of the sequence of interest,for example, by restriction enzyme digests or PCR.

[0386] To prepare a larger quantity of viral vector, 2 to 5 microgramsof destination vector comprising the sequence of interest may bedigested with Pacd restriction enzyme to expose the adenoviral ITRs(immediately adjacent to the Pacd sites on the 5′ and 3′ ends of theadenoviral genome). The digested DNA may be purified using anyconventional technique, for example, phenol/chloroform extractionfollowed by ethanol precipitation, or use of a commercially availablekit for this purpose.

[0387] The digested DNA is used to transfect an appropriate host cell,for example, 293 cells. The day before transfection, 6 well plates with5×10 ⁵293 cells per well may be prepared. On the day of transfection, 2micrograms of DNA is used to transfect the cells in each well.Transfection may be accomplished using standard techniques using, forexample, calcium phosphate, lipids, electroporation, etc. Preferredmethods of transfection include those utilizing cationic lipids ormixtures of cationic and neutral lipids. Suitable transfection reagentsare commercially available, for example, from Invitrogen Corporation,Carlsbad, Calif. One suitable lipid formulation is Lipofectamine™ 2000.

[0388] The day after transfection, the transfection media may be removedand replaced with fresh media. The next day, the transfected cells maybe trypsinized and transferred. The cells from one well are used to seeda 100 mm dish. The cells are grown in the 100 mm dish for 7-10 days. Themedia is replaced with fresh media every 2-3 days. At about 9 days posttransfection, “plaques” may be observed forming in the monolayer of 293cells. Plaques will appear as cleared areas when viewed by the nakedeye. Under the microscope, plaques will be fringed with rounded, lysingcells. This is referred to as cytopathic effect (CPE). The media shouldbe replaced with fresh media every 2 days until most of the cells aredemonstrating CPE.

[0389] Harvest the plate by squirting off the cells using the growthmedia and transfer the cells and media to a 15 ml tube. Freeze/thaw thetube 3 times by alternating −80° C. and 37° C. This releases the viralparticles from the cells. Centrifuge the tube to remove the unwantedcellular debris (3000 rpm×10 minutes). Remove the supernatant andtransfer it to a fresh tube. This material now contains recombinantadenoviral vector containing the sequence of interest. This can be useddirectly in experiments to deliver the sequence of interest.

[0390] To increase the titer of the viral vector, the viral vector maybe amplified, for example, by applying a small amount (typically 100microliters) of the initial viral vector to a fresh plate of 293 cells(typically 5×10⁶ 293 cells in a 100 mm dish). Infection of the cellsoccurs within the first couple hours and three days later CPE isobserved throughout the plate. Viral vector is harvested as describedabove.

[0391] Viral vector produced in this way (called “crude viral lysates”,or CVLs) is typically high titer (>10⁹ infectious virus/ml) and can beused directly for most applications. To determine the exact titer of theCVL (or of any adenoviral stock), 293 cells are plated at 1×10⁶ cellsper well in 6-well plates. The next day, each well is transduced with 1ml media containing ten-fold serial dilutions of CVL ranging from 10⁻⁵to 10⁻¹⁰. After overnight incubation, the media is removed and the cellmonolayers are overlaid with 2 ml of fresh media containing 0.4%Ultrapure agarose. This semi-solid medium prevents viral vector fromdiffusing throughout the plate and keeps individual plaques distinct.After 7 to 10 days, distinct plaques will be visible to the naked eye.MTT (3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl-tetrazolium bromide) canbe used to stain the wells to aid in plaque visualization. Plaques arecounted, and that number is multiplied by the dilution factor to obtainthe titer of infectious viral vector present in the original CVL. Ifhigher titer viral vector is required, the viral vector in the CVLs canbe concentrated and purified using a number of different approachesincluding: cesium chloride density ultracentrifugation, HPLC, orcommercially available columns designed for virus purification (e.g.Virapur). These methods typically result in titers of >11 infectiousvirus/ml.

EXAMPLE 2 Use of Suppressor tRNAs to Generate Fusion Polypeptides

[0392] Detection of expressed polypeptides is often facilitated by theuse of epitope tags (e.g. V5 or myc) or detectable markers (e.g.,β-lactamase, β-galactosidase, β-glucuronidase, GFP, etc.). This isespecially useful if there is no specific antibody available for thepolypeptide of interest. However, addition of epitope tags and/or fusionto a detectable marker may adversely affect polypeptide activity,structure, or its interaction with other molecules. One common approachto this problem is to clone the gene of interest twice: with and withoutthe tag.

[0393] The present invention provides materials and methods to express apolypeptide with and without a tag or marker from the same geneticconstruct. This is accomplished using mammalian suppressor tRNAs thatspecifically recognize and decode one of the three stop codons (Ochre,Amber, and Opal) and result in the insertion of an amino acid at theposition coded for by the stop codon. The suppressor tRNAs may insertany amino acid into the position coded for by the stop codon. In thespecific embodiments described below, the amino acid serine wasinserted; however, any amino acid desired can be inserted by preparingand expressing the appropriate suppressor tRNA according to the presentinvention.

[0394] Expression plasmids encoding a reporter gene with all threepossible stop codons in frame with C-terminal tags were constructed.Following delivery of suppressor tRNAs in trans, the stop codons betweenthe gene and the epitope tag were suppressed, allowing translation ofthe 3′ sequences.

[0395] Plasmids encoding each suppressor tRNA were co-transfected withthe corresponding expression plasmid to test the efficiency ofsuppression. Suppression of TAA and TAG were approximately 50% to 60%efficient, while TGA was only 30%. Changing the nucleotide following theTGA stop codon from an adenine to a cytosine improved suppression toabout 70%.

[0396] A recombinant adenoviral vector was constructed that expresses asuppressor tRNA. A map of a plasmid containing the adenoviral constructpAd-GW-TO/tRNA in which a suppressor tRNA is under the control of atetracycline-inducible CMV promoter is shown in FIG. 7. The nucleotidesequence of pAd-GW-TO/tRNA is provided in Table 7 (SEQ ID NO: ). Anadditional adenoviral construct expressing a suppressor tRNA ispAdenoTAG tRNA shown in FIG. 8. The nucleotide sequence of pAdenoTAGtRNA is provided in Table 8. Table 9 provides the nucleotide sequence ofa Sau3A fragment that may be used to construct suppressor tRNAcontaining nucleic acid molecules of the invention (e.g., pAdenoTagtRNA.) A transcription terminator is located at bases 600 to 606 of thefragment, the sequence corresponding to the suppressor tRNA is locatedat bases 512 to 593 of the fragment, the anti-codon is located at bases545 to 547, and the tetracycline operator sequence is located at bases474 to 511. The suppressor tRNA produced from this sequence suppressesthe amber stop codon UAG. Those skilled in the art will appreciated thatit is possible to prepare suppressors for opal and ochre stop codons bymutating the bases in the anti-codon to make the anti-codon the reversecomplement of the stop codon. i.e., TCA for the opal stop codon and TTAfor the ochre stop codon. Other anti-codons may be used, for example,those employing other bases in the wobble position. Constructing asuitable sequence from which to produce a desired suppressor tRNA (e.g.,by introducing one or more point mutations in a sequence) is routine inthe art.

[0397] The plasmid may be digested with Pacd to generate an infectiousadenoviral genome. The viral vector expressing the suppressor tRNA maybe used in conjunction with any vector comprising a sequence with a stopcodon to be suppressed. In some embodiments, a viral vector expressing asuppressor tRNA and a viral vector comprising a sequence of interest maybe used to co-infect a cell and produce a fusion polypeptide. A fusionpolypeptide may be encoded entirely by the sequence of interest, forexample, the sequence may have one open reading frame (ORF) separatedfrom another ORF by a stop codon. Alternatively, one ORF may be presenton the sequence of interest and one or more additional ORFs may bepresent on the viral vector. Co-infection with a suppressor-expressingviral vector an expression vector will result in the expression of afusion polypeptide; infection without the suppressor-expressing viralvector will produce a native polypeptide. Thus, the suppressiontechnology allows expression of tagged and untagged polypeptides using asingle expression vector.

EXAMPLE 3 Detailed Materials and Method for Construction of AdenoviralVectors and Kits

[0398] Kits of the invention may comprise one or more sets ofinstructions for carrying out the methods of the invention. For example,the instructions may related to the propagation of cells used in themethods of the invention and/or to conducting individual reactions thatare part of the methods. In a one embodiments, kits of the invention maycomprise instructions for growth and maintenance of cell used in methodsof the invention (e.g., the 293A cell line manual, catalog no. R705-07version B, Invitrogen Corporation, Carlsbad, Calif.) and a manual forthe preparation of the viral vectors of the invention (e.g., theViraPower™ Adenoviral Expression System manual, catalog no. K4930-00,version A, Invitrogen Corporation, Carlsbad, Calif.).

[0399] In one embodiment, a kit of the invention may comprise thenecessary reagents and instructions to prepare a viral vector accordingto the invention. Such a kit may comprise one or more componentsselected from the group consisting of: the ViraPower™ AdenoviralGATEWAY™ Expression Kit, ViraPower™ Adenoviral Promoterless GATEWAY™Expression Kit, pAd/CMV/V5-DEST™ GATEWAY™ Vector Pack, or pAd/PL-DEST™GATEWAY™ Vector Pack all available from Invitrogen Corporation,Carlsbad, Calif.

[0400] A plasmid map of pAd/PL-DEST™ is provided in FIG. 9 and thesequence of the plasmid is provided in Table 10.

[0401] A kit may also comprise one or more control reagents. Forexample, a kit may comprise an adenoviral vector comprising a detectablemarker that may be used as a control for transfection of cells andinfection of cells. One suitable control reagent is pAd/CMV/V5-GW/lacZcontrol. A map of the pAd/CMV/V5-GW/lacZ plasmid is provide as FIG. 10and the nucleotide sequence of the plasmid is provided as Table 11.

[0402] Kits of the invention may comprise one or more additionalproducts (e.g., accessory products). Such products include, but are notlimited to, reagents and materials for purifying nucleic acids (e.g.,plasmid purification), host cells for propagating plasmids and/orviruses (e.g., E. coli and 293 cells), transfection reagents (e.g.,lipids), reagents for assaying control vector expression (e.g.,β-lactamase assay reagents, β-galactosidase assay reagents, antibodiesto β-galactosidase), recombination polypeptides, and antibiotics forselection of transformed cells. The contents of one suitable kitinclude, ViraPower™ Adenoviral GATEWAY™ Expression Kit, ViraPower™Adenoviral Promoterless GATEWAY™ Expression Kit, 293A Cell Line,GATEWAY™ LR Clonase™ Enzyme Mix, Library Efficiency® DB3.1™ CompetentCells, One Shot® TOP10 Chemically Competent E. coli, S.N.A.P.™ MidiPrepKit, Lipofectamine™ 2000, β-gal Antiserum, and Ampicillin all availablefrom Invitrogen Corporation, Carlsbad, Calif.

[0403] A polypeptide encoded by a sequence of interest may be expressedas a fusion polypeptide with a detectable epitope. For example, apolypeptide expressed from pAd/CMV/V5-DEST™ (FIG. 6), can be detectedwith an antibody to the V5 epitope. Antibodies to the detectable epitopemay be labeled, for example, horseradish peroxidase (HRP) or alkalinephosphatase (AP) may be conjugated to the antibody to allow one-stepdetection using chemiluminescent or colorimetric detection methods. Afluorescent label, (e.g., FITC) may be conjugated to the antibody toallow one-step detection in immunofluorescence experiments. Thus, kitsof the invention may comprise one or more antibodies to one or moredetectable epitopes. Antibodies to detectable epitopes may be labeled.Suitable antibodies include, but are not limited to, an anti-V5antibody, an anti-V5-HRP antibody, an anti-V5-AP antibody, and/or ananti-V5-FITC antibody.

[0404] Examples of nucleic acid molecules of the invention includepAd/CMV/V5-DEST™ (36.7 kb) and pAd/PL-DEST™ (34.9 kb), which aredestination vectors adapted for use with recombinational cloning (e.g.,GATEWAY™ Technology), and are designed to allow high-level, transientexpression of recombinant fusion polypeptides in dividing andnon-dividing mammalian cells, for example, using ViraPower™ AdenoviralExpression System, catalog nos. K4930-00 and K4940-00 available fromInvitrogen Corporation, Carlsbad, Calif.

[0405] A choice of vectors permits the construction of an adenovirusexpressing a sequence of interest. Each vector provides differentfeatures that may be useful under different circumstances. For example,the pAd/CMV/V5-DEST™ vector contains the CMV promoter that provideshigh-level, constitutive expression of the sequence of interest and theC-terminal V5 epitope for detection of recombinant polypeptide usinganti-V5 antibodies. The pAd/PL-DEST™ vector has no promoter allowingexpression of a sequence of interest from any desired promoter that maybe operably linked to the sequence of interest, optionally, prior toinsertion in the viral vectors of the invention. Additionally, thepAd/PL-DEST™ vector has no 3′ sequences allowing addition of aC-terminal epitope tag (if desired) and a polyadenylation signal ofchoice.

[0406] The pAd/CMV/V5-DEST™ vector (36686 bp) contains the followingfeatures. Feature Benefit Human adenovirus type 5 Encodes all elements(except E1 sequences (corresponds to and E3 polypeptides) required towild-type 1-458 and produce replication-incompetent 3513-35935 sequence)adenovirus (Russell, (2000) J. Gen. Note: The E1 and E3 regions Virol.81, 2573-2604.) are deleted. including: Left and right ITRsEncapsidation signal for packaging E2 and E4 regions Late genes pAdforward priming Permits sequencing of the site insert. CMV promoterPermits high-level expression of the gene of interest T7promoter/priming Allows in vitro transcription in site the senseorientation and sequencing through the insert. attR1 and attR2 sitesBacteriophage λ-derived DNA recombination sequences that permitrecombinational cloning of the gene of interest from a GATEWAY ™ entryclone. ccdB gene Permits negative selection of the plasmid.Chloramphenicol Allows counterselection of the resistance gene (Cm^(R))plasmid. V5 epitope Allows detection of the recombinant fusionpolypeptide by the Anti-V5 Antibodies Herpes Simplex Virus Permitsefficient transcription thymidine kinase (TK) termination andpolyadenylation ° polyadenylation signal of mRNA pAd reverse primingsite Allows sequencing of the insert in the anti-sense orientation. pUCorigin Permits high-copy replication and maintenance in E. coli. blapromoter Allows expression of the ampicillin resistance gene. Ampicillinresistance gene Allows selection of the plasmid (β-lactamase) in E.coli. Pac I restriction sites Permits exposure of the left and(positions 34610 and 36684) right ITRs required for viral replicationand packaging.

[0407] The pAd/PL-DEST™ vector (34864 bp) contains the followingfeatures. Feature Benefit Human adenovirus type 5 Encodes all elements(except E1 sequences (corresponds and E3 proteins) required to towild-type 1-458 produce replication-incompetent and 3513-35935adenovirus (Russell, 2000) sequence) including: Note: The E1 and E3 Leftand right ITRs regions are deleted. Encapsidation signal for packagingE2 and E4 regions Late genes pAd forward priming site Permits sequencingof the insert. attR1 and attR2 sites Bacteriophage λ-derived DNArecombination sequences that permit recombinational cloning of the DNAsequence of interest from a GATEWAY ™ entry clone (Landy, 1989, Annu.Rev. Biochem. 58, 913-949.). Chloramphenicol Allows counterselection ofthe resistance gene (Cm^(R)) plasmid. ccdB gene Permits negativeselection of the plasmid. pAd reverse priming site Allows sequencing ofthe insert in the anti-sense orientation. pUC origin Permits high-copyreplication and maintenance in E. coli. bla promoter Allows expressionof the ampicillin resistance gene. Ampicillin resistance Allowsselection of the plasmid gene (β-lactamase) in E. coli. Pac Irestriction sites Permits exposure of the left and (positions 32788 and34862) right ITRs required for viral replication and packaging.

[0408] The pAd/CMV/V5-DEST™ and pAd/PL-DEST™ vectors contain thefollowing features: human adenovirus type 5 sequences (Ad 1-458),upstream of the attR1 site, containing the “Left” Inverted TerminalRepeat (L-ITR) and the encapsidation signal sequence required for viralpackaging; human cytomegalovirus (CMV) immediate early promoter forhigh-level constitutive expression of the gene of interest in a widerange of mammalian cells (in pAd/CMV/V5-DEST™ only; (Andersson, et al.,1989, J. Biol. Chem. 264, 8222-8229; Boshart, et al., 1985, Cell 41,521-530; Nelson, et al., 1987, Molec. Cell. Biol. 7, 4125-4129); tworecombination sites, attR1 and attR2 for recombinational cloning of theDNA sequence of interest from an entry clone; chloramphenicol resistancegene (Cm^(R)) located between the two attR sites for counterselection;the ccdB gene located between the attR sites for negative selection;C-terminal V5 epitope for detection of the recombinant polypeptide ofinterest (in pAd/CMV/V5-DEST™ only); (Southern, et al., 1991, J. Gen.Virol. 72, 1551-1557); human adenovirus type 5 sequences (Ad 3513-35935)containing genes and elements (e.g. E2 and E4 regions, late genes, and“Right” ITR) required for proper packaging and production of adenovirus(Hitt, et al., (1999) In The Development of Human Gene Therapy, T.Friedmann, ed. (Cold Spring Harbor, N.Y.: Cold Spring Harbor LaboratoryPress), pp. 61-86.; Russell, (2000)); ampicillin resistance gene forselection in E. coli; and the pUC origin for high-copy replication andmaintenance of the plasmid in E. coli. In one alternative of this aspectof the invention, the chloramphenicol resistance gene in the cassettecan be replaced by a spectinomycin resistance gene (see Hollingshead etal., Plasmid 13(1):17-30 (1985), NCBI accession no. X02340 M10241), andthe destination vector containing attP sites flanking the ccdB andspectinomycin resistance genes can be selected onampicillin/spectinomycin-containing media. It has recently been foundthat the use of spectinomycin selection instead of chloramphenicolselection results in an increase in the number of colonies obtained onselection plates, indicating that use of the spectinomycin resistancegene may lead to an increased efficiency of cloning from that observedusing cassettes containing the chloramphenicol resistance gene.

[0409] The plasmid, pAd/CMV/V5-GW/lacZ, is included and may be used as apositive expression control in the mammalian cell line of choice.pAd/CMV/V5-GW/lacZ (FIG. 10) is a 37567 bp vector expressingβ-galactosidase, and was generated using the GATEWAY™ LR recombinationreaction between an entry clone containing the lacZ gene andpAd/CMV/V5-DEST™. β-galactosidase is expressed as a C-terminal V5 fusionpolypeptide with a molecular weight of approximately 120 kDa.

[0410] Nucleic acid molecules of the invention may be constructed usingany technique known to those skilled in the art, for examplerecombinational cloning (e.g., using GATEWAY™). GATEWAY™ is a universalcloning technology that takes advantage of the site-specificrecombination properties of bacteriophage lambda (Landy, 1989) toprovide a rapid and highly efficient way to move a DNA sequence ofinterest into multiple vector systems. To express a sequence of interestin mammalian cells using the GATEWAY™ Technology the following methodmay be used. First, a sequence of interest may be cloned into a GATEWAY™entry vector of choice to create an entry clone. If pAd-DEST™ is used, apromoter of choice and a polyadenylation signal may be operably attachedto the sequence of interest. Next, a recombination reaction (e.g., an LRreaction) may be performed to generate an expression clone bytransferring the sequence of interest into a GATEWAY™ destination vector(e.g. pAd/CMV/V5-DEST or pAd-DEST™). An expression clone may then beused to generate viral vector using the ViraPower™ Adenoviral ExpressionSystem.

[0411] For more information about the GATEWAY™ Technology, generating anentry clone, and performing the LR recombination reaction, refer to theGATEWAY™ Technology manual.

[0412] Materials and methods of the invention (e.g., The ViraPower™Adenoviral Expression System) facilitate highly efficient, in vitro orin vivo delivery of a target gene to dividing and non-dividing mammaliancells using a replication-incompetent adenovirus. The System utilizesGATEWAY™-adapted destination vectors to allow highly efficient and rapidcreation of adenoviral vectors that circumvent the need for traditional,homologous recombination and the use of recA⁺ bacteria to produceadenovirus. To express a sequence of interest in mammalian cells usingthe ViraPower™ Adenoviral Expression System the following method may beused. First, an expression clone in pAd/CMV/V5-DEST™ or pAd-DEST™ may becreated (e.g., using GATEWAY™ Technology or other suitable methodology).Next, the expression clone may be digested with Pac I to expose theviral inverted terminal repeats (ITRs). The digested expression clonemay be introduced into suitable host cells (e.g., 293 or 293A cells) toproduce adenovirus. The adenovirus may be amplified by infectingadditional cells and allowing the virus to replicate. The virus may beused to transduce a suitable cell line (e.g., a mammalian cell line ofchoice). The transduced cell line may be assayed for expression of thesequence of interest using any suitable means.

[0413] The pAd/CMV/V5-DEST™ and pAd/PL-DEST™ vectors may be linear ormay be supercoiled plasmids. Each destination vector may be supplied as6 μg of plasmid, lyophilized in TE, pH 8.0. To use, resuspend thedestination plasmid in 40 μl of sterile water to a final concentrationof 150 ng/μl.

[0414] It may be desirable to propagate and maintain thepAd/CMV/V5-DEST™ and pAd/PL-DEST™ vectors. One suitable method is to useLibrary Efficiency® DB3.1™ Competent Cells (Invitrogen Corporation,Carlsbad, Calif.) for transformation. The DB3.1™ E. coli strain isresistant to CcdB effects and can support the propagation of plasmidscontaining the ccdb gene. To maintain integrity of the vector, selectfor transformants in media containing 50-100 μg/ml ampicillin and 15-30μg/ml chloramphenicol. General E. coli cloning strains including TOP10or DH5α are not recommended for propagation and maintenance as thesestrains are sensitive to CcdB effects.

[0415] To recombine a sequence of interest into pAd/CMV/V5-DEST™ orpAd-DEST™, the sequence of interest should be cloned into an entryclone. Many entry vectors including pENTR/D-TOPO® are available fromInvitrogen Corporation, Carlsbad, Calif. to facilitate generation ofentry clones.

[0416] pAd/CMV/V5-DEST™ is a C-terminal fusion vector; however, thisvector may be used to express native polypeptides or C-terminal fusionpolypeptides. A sequence of interest encoding a polypeptide of interestmust contain an ATG initiation codon in the context of a Kozak consensussequence for proper initiation of translation in mammalian cells (Kozak,M. (1987). Nucleic Acids Res. 15, 8125-8148. Kozak, M. (1991). J. CellBiology 115, 887-903. Kozak, M. (1990). Proc. Natl. Acad. Sci. USA 87,8301-8305.). An example of a Kozak consensus sequence is (G/A)NNATGG(SEQ ID NO:). The ATG initiation codon is underlined. Note that othersequences are possible, but the G or A at position −3 and the G atposition +4 are the most critical for function (shown in bold).

[0417] If it is desired to include the V5 epitope tag, a sequence ofinterest in the entry clone should not contain a stop codon. Inaddition, the sequence encoding the polypeptide should be in frame withthe V5 epitope tag after recombination. To express a native polypeptide(e.g., without a tag sequence) from a sequence of interest, the sequenceof interest must contain a stop codon in the entry clone. The C-terminalpeptide containing the V5 epitope and the attB2 site will addapproximately 4.3 kDa to the size of a polypeptide expressed from asequence of interest.

[0418] pAd/PL-DEST™ allows generation of an adenovirus that contains asequence of interest whose expression is controlled by a promoter ofchoice. To facilitate proper expression of a sequence of interest frompAd/PL-DEST™, an entry clone containing the following should begenerated: 1) a promoter of choice to control expression of the sequenceof interest in mammalian cells; 2). the sequence of interest; 3) a stopcodon; and 4) a polyadenylation signal sequence of choice for propertranscription termination and polyadenylation of mRNA. To express apolypeptide from a sequence of interest, the ORF of the polypeptideshould contain an ATG initiation codon in the context of a Kozakconsensus sequence for proper initiation of translation in mammaliancells (Kozak, 1987; Kozak, 1991; Kozak, 1990). If desired, an N-terminaland/or C-terminal fusion tag sequence may be included.

[0419] In some embodiments, an entry clone contains attL sites flankingthe sequence of interest. Sequences of interest in an entry clone aretransferred to the destination vector backbone by mixing the DNAs withthe GATEWAY™ LR Clonase™ Enzyme Mix, Invitrogen Corporation, Carlsbad,Calif. The resulting LR recombination reaction is then transformed intoE. coli (e.g. TOP10 or DH5α™-T1^(R)) and the expression clone selectedusing ampicillin. Recombination between the attR sites on thedestination vector and the attL sites on the entry clone replaces thechloramphenicol (Cm^(R)) gene and the ccdB gene with the sequence ofinterest and results in the formation of attB sites in the expressionclone.

[0420] The ccdb gene mutates at a very low frequency, resulting in avery low number of false positives. True expression clones will beampicillin- and blasticidin-resistant and chloramphenicol-sensitive.Transformants containing a plasmid with a mutated ccdB gene will beampicillin-, blasticidin-, and chloramphenicol-resistant. To check aputative expression clone, test for growth on LB plates containing 30μg/ml chloramphenicol. A true expression clone should not grow in thepresence of chloramphenicol.

[0421] The recombination region of the expression clone resulting frompAd/CMV/V5-DEST™×entry clone is shown in FIG. 8. Shaded regionscorrespond to those DNA sequences transferred from the entry clone intothe pAd/CMV/V5-DEST™ vector by recombination. Non-shaded regions arederived from the pAd/CMV/V5-DEST™ vector. Bases 1414 and 3657 of thepAd/CMV/V5-DEST™ sequence are marked. The recombination region of theexpression clone resulting from pAd/PL-DEST™×entry clone is shown INFIG. 9. Shaded regions correspond to those DNA sequences transferredfrom the entry clone into the pAd/PL-DEST™ vector by recombination.Non-shaded regions are derived from the pAd/PL-DEST™ vector. Bases 519and 2202 of the pAd/PL-DEST™ sequence are marked.

[0422] To confirm that a sequence of interest is in the correctorientation and in frame with a fusion tag (if present), an expressionconstruct may be sequenced. The following primer binding may be used tosequence an expression construct. Refer to the FIGS. 8 and 9 for thelocation of the primer binding sites. The pAd/CMV/V5-DEST™ vectorcontains the T7 promoter/priming site 5′-TAATACGACTCACTATAGGG-3′ (SEQ IDNO:) and the V5 (C-term) reverse priming site5′-ACCGAGGAGAGGGTTAGGGAT-3′ (SEQ ID NO:). The pAd/PL-DEST™ vectorcontains the pAd forward priming site 5′GACTTTGACCGTTTACGTGGAGAC-3′ (SEQID NO:) and the pAd reverse priming site 5′-CCTTAAGCCACGCCCACACATTTC-3′(SEQ ID NO:).

[0423] Once purified plasmid DNA of a pAd/CMV/V5-DEST™ or pAd/PL-DEST™expression construct has been obtained, the vector may be used inViraPower™ Adenoviral Expression System (Invitrogen Corporation,Carlsbad, Calif.) by digesting with Pac I. The Pac I-digested vector isused to produce an adenoviral stock, which after amplification, may thenbe used to transduce a mammalian cell line of choice to express thesequence of interest or a polypeptide encoded by the sequence ofinterest.

[0424] Once a pAd/CMV/V5-DEST and/ or a pAd/PL-DEST™ expression clonehas been constructed, purified plasmid DNA may be prepared. Suitablepurification methods include the S.N.A.P.™ MidiPrep Kit (InvitrogenCorporation, Carlsbad, Calif.) and CsCl gradient centrifugation. Toverify the integrity of an expression construct after plasmidpreparation, the plasmid may be analyzed by restriction digests.

[0425] Before transfecting an expression clone into 293A cells, the leftand right viral ITRs on the vector should be exposed to allow properviral replication and packaging. Both pAd/CMV/V5-DEST™ and pAd/PL-DEST™vectors contain Pac I restriction sites. Digestion of the vector withPac I allows exposure of the left and right viral ITRs and removal ofthe bacterial sequences (i.e. pUC origin and ampicillin resistancegene). The sequence of interest must not contain any Pac I restrictionsites.

[0426] Digest at least 5 μg of purified plasmid DNA of pAd/CMV/V5-DEST™or pAd/PL-DEST expression construct with Pac I using commerciallyavailable Pac I enzyme. Follow the manufacturer's instructions. Purifythe digested plasmid DNA using phenol/chloroform extraction followed byethanol precipitation or a DNA purification kit (e.g. S.N.A.P. MiniPrep™Kit, catalog no. K19001, Invitrogen Corporation, Carlsbad, Calif.). Gelpurification is not required.

[0427] Resuspend or elute the purified plasmid, as appropriate insterile water or TE Buffer, pH 8.0 to a final concentration of 0.1-3.0μg/μl.

[0428] To express a gene of interest from pAd/CMV/V5-DEST™ orpAd/PL-DEST™ using Invitrogen's ViraPower™ Adenoviral Expression System,the following reagents are required: 1) a host cell (e.g., 293 or 293Acell lines); and 2) a transfection reagent (e.g., Lipofectamine™ 2000Reagent, catalog no. 11668019, Invitrogen Corporation, Carlsbad,Calif.). The 293A cell line is a subclone of the 293 cell line andsupplies the E1 proteins required for production ofreplication-competent adenovirus and exhibits a flattened morphology toenhance visualization of plaques.

[0429] pAd/CMV/V5-GW/lacZ is included with the each kit for use as apositive control for expression in the ViraPower™ Adenoviral ExpressionSystem. In pAd/CMV/V5-GW/lacZ, β-galactosidase is expressed as aC-terminally tagged fusion polypeptide that may be easily detected bywestern blot or functional assay. To propagate and maintain the plasmid:resuspend the vector in 10 μl of sterile water to prepare a 1 μg/μlstock solution. Use the stock solution to transform a recA, endA E. colistrain like TOP 10, DH5α™-T1^(R), or equivalent. Use 10 ng of plasmidfor transformation. Select transformants on LB agar plates containing50-100 μg/ml ampicillin. Prepare a glycerol stock of a transformantcontaining plasmid for long-term storage.

EXAMPLE 4 Exemplary Instruction Manual for Kits of the Invention.

[0430] Provided for in the methods of the present invention is a kitcontaining a viral system for high-level, transient expression individing and non-dividing mammalian cells. One nonlimiting example ofsuch a kit is the ViraPower™ Adenoviral Expression System, Invitrogencatalog nos. K4930-00 and K4940-00, Version A, Jul. 15, 2002, 25-0543,as described in this example.

[0431] The ViraPower™ Adenoviral Expression Kits include the followingcomponents. For a detailed description of the contents of eachcomponent, see below. Catalog No. Catalog No. Components K4930-00K4940-00 pAd/CMV/V5-DEST ™ ✓ GATEWAY ™ Vector pAd/PL-DEST ™ GATEWAY ™ ✓Vector 293A Cell Line ✓ ✓

[0432] The ViraPower™ Adenoviral Expression Kits are shipped asdescribed below. Upon receipt, store each component as detailed below.Item Shipping Storage pAd-DEST ™ GATEWAY ™ Vector Blue ice −20° C. 293ACell Line Dry ice Liquid nitrogen

[0433] Each ViraPower™ Adenoviral Expression Kit includes a destinationvector (pAd/CMV/V5-DEST™ or pAd/PL-DEST™) for cloning a DNA sequence ofinterest and a corresponding expression control vector. For informationabout the vectors see, for example, the pAd/CMV/V5-DEST™ andpAd/PL-DEST™ GATEWAY™ Vector manual, catalog nos. V493-20 and 494-20,version B, Invitrogen Corporation, Carlsbad, Calif.

[0434] Methods of the invention may be practiced using any suitable cellline (e.g., 293A Cell Line, catalog no. R-705-07, InvitrogenCorporation, Carlsbad, Calif.).

[0435] A number of reagents that are commercially available may be usedin conjunction with the methods of the invention. For example, thefollowing reagents may be obtained from Invitrogen Corporation,Carlsbad, Calif. Item Catalog no. pAd/CMV/V5-DEST ™ GATEWAY ™ VectorV493-20 pAd/PL-DEST ™ GATEWAY ™ Vector V494-20 293A Cell Line R705-07Lipofectamine ™ 2000 11668-027 11668-019 Opti-MEM ® I Reduced SerumMedium 31985-062 31985-062 Phosphate-Buffered Saline (PBS), pH 7.410010-023 10010-031 S.N.A.P ™ MidiPrep Kit K1910-01

[0436] The ViraPower™ Adenoviral Expression System allows creation of areplication-incompetent adenovirus that can be used to deliver andexpress a gene of interest in either dividing or non-dividing mammaliancells. The major components of the ViraPower™ Adenoviral ExpressionSystem include: a choice of GATEWAY™-adapted adenoviral vectors thatallow highly efficient generation of a recombinant adenovirus containingthe gene of interest under the control of the human cytomegalovirus(CMV) immediate-early enhancer/promoter (pAd/CMV/V5-DEST™) or a promoterof choice (pAd/PL-DEST™); a optimized cell line, 293A, which allowsproduction and subsequent, titering of the recombinant adenovirus; and acontrol expression plasmid containing the lacZ gene which, when packagedinto virions and transduced into a mammalian cell line, expressesβ-galactosidase. For more information about the adenoviral vectors, thecorresponding positive control vector containing the lacZ gene, andGATEWAY™ Technology, refer to the pAd/CMV/V5-DEST™ and pAd/PL-DEST™GATEWAY™ Vectors manual. This manual is supplied with each ViraPower™Adenoviral Expression Kit, but may also be obtained by contactingInvitrogen Corporation, Carlsbad, Calif.

[0437] Use of the ViraPower™ Adenoviral Expression System to facilitateDNA virus-based expression of the gene of interest provides thefollowing advantages: uses GATEWAY™ Technology to allow highlyefficient, rapid cloning of a gene of interest into a full-lengthadenoviral vector, bypassing the need for a shuttle vector andinefficient homologous recombination in human or bacterial cells; allowsgeneration of high titer adenoviral stocks (i.e., 1×10⁹ pfu/ml in crudepreparations and 1×10¹¹ pfu/ml in concentrated preparations);efficiently delivers the gene of interest to actively dividing andnon-dividing mammalian cells in culture or in vivo; generates adenoviralconstructs with such a high degree of efficiency and accuracy that thesystem is amenable for use in high-throughput applications or librarytransfer procedures; and allows production of a replication-incompetentvirus that enhances the biosafety of the system and its use as a genedelivery vehicle.

[0438] This example provides an overview of the ViraPower™ AdenoviralExpression System and provides instructions and guidelines to: transfectthe pAd/CMV/V5-DEST™ or pAd/PL-DEST™ expression construct into the 293ACell Line to produce an adenoviral stock; amplify the adenoviral stock;titer the adenoviral stock; use the amplified adenoviral stock totransduce any mammalian cell line of choice; and assay for transientexpression of any polynucleotide of interest or recombinant polypeptide.This expression may be used to express, for example, a polypeptide, aprotein, or an untranslated RNA, e.g., tRNA, all of which areencompassed by the term “gene of interest” as used herein.

[0439] For details and instructions to generate an expression constructusing pAd/CMV/V5-DEST™ or pAd/PL-DEST™, refer to the pAd/CMV/V5-DEST™ orpAd/PL-DEST™ GATEWAY™ Vector manual. For instructions to culture andmaintain the 293A producer cell line, refer to the 293A Cell Linemanual. These manuals are supplied with the ViraPower™ AdenoviralExpression Kits, and are also available from Invitrogen Corporation,Carlsbad, Calif.

[0440] The ViraPower™ Adenoviral Expression System facilitates highlyefficient, in vitro or in vivo delivery of a target gene to dividing andnon-dividing mammalian cells using a replication-incompetent adenovirus.Based on the second-generation vectors developed by Bett, A.J., et al.,Proc. Natl. Acad. Sci. USA 91:8802-8806 (1994), the ViraPower™Adenoviral Expression System takes advantage of the GATEWAY™ Technologyto simplify and greatly enhance the efficiency of generating high-titer,recombinant adenovirus.

[0441] The first major component of the system described in this exampleis an E1 and E3-deleted, pAd-DEST™-based expression vector into whichthe gene of interest will be cloned. Expression of the gene of interestis controlled by the human cytomegalovirus (CMV) promoter (inpAd/CMV/V5-DEST™) or the promoter of choice (in pAd/PL-DEST™). Thevector also contains the elements required to allow packaging of theexpression construct into virions (e.g., 5′ and 3′ ITRs, encapsidationsignal, adenoviral late genes). For more information about the pAd-DEST™expression vectors, refer to the pAd/CMV/V5-DEST™ and pAd/PL-DEST™GATEWAY™ Vector manual, available from Invitrogen Corporation, Carlsbad,Calif.

[0442] The second major component of the system is an optimized 293ACell Line that will be used to facilitate initial production,amplification, and titering of replication-incompetent adenovirus. The293A cells contain a stably integrated copy of E1 that supplies the E1proteins (E1 a and E1 b) in trans that are required to generateadenovirus. For more information about the 293A Cell Line, refer to the293A Cell Line manual, available from Invitrogen Corporation, Carlsbad,Calif. The pAd-DEST™ vector containing the gene of interest istransfected into 293A cells to produce a replication-incompetentadenovirus. The crude adenoviral stock is used to infect 293A cells toproduce an amplified adenoviral stock. Once the adenoviral stock isamplified and titered, this high-titer stock may be used to transducethe recombinant adenovirus into the mammalian cell line of choice forexpression of the recombinant polypeptide of interest.

[0443] Adenovirus enters target cells by binding to theCoxsackie/Adenovirus Receptor (CAR). After binding to the CAR, theadenovirus is internalized via integrin-mediated endocytosis followed byactive transport to the nucleus. Once in the nucleus, the early eventsare initiated (e.g., transcription and translation of E1 proteins),followed by expression of the adenoviral late genes and viralreplication. Expression of the late genes is dependent upon E1. In theViraPower™ Adenoviral Expression System, E1 is supplied by the 293Aproducer cells. The viral life cycle spans approximately 3 days. Formore information about the adenovirus life cycle and adenovirus biology,refer to the following references as well as published reviews:Bergelson, J. M., et al. Science 275:1320-1323 (1997); Hitt, M.M., etal., “Structure and Genetic Organization of Adenovirus Vectors,” in TheDevelopment of Human Gene Therapy, Friedmann, T., ed., Cold SpringHarbor Laboratory Press, Cold Spring Harbor, N.Y. (1999), pp. 61-86.

[0444] After adenovirus is transduced into the target cell and istransported to the nucleus, it does not integrate into the host genome.Therefore, expression of the gene of interest is typically detectablewithin 24 hours after transduction and is transient, only persisting foras long as the viral genome is present. Additional information regardingthe use of adenoviral vectors and host cells may be obtained from thefollowing references: Bett, A. J., et al., Proc. Natl. Acad. Sci. USA91:8802-8806 (1994); Chen, H. H., et al., Hum. Gene Ther. 10:365-373(1999); Ciccarone, V., et al., Focus 21:54-55 (1999); Dion, L. D., etal., J. Virol. Methods 56:99-107 (1996); Engelhardt, J. F., et al.,Nature Genetics 4:27-34 (1993); Fallaux, F. J., et al., Hum. Gene Ther.9:1909-1917 (1998); Fallaux, F. J., et al., Hum. Gene Ther. 7:215-222(1996); Fan, X., et al, Hum. Gene Ther. 11:1313-1327 (2000); Graham, F.L., et al., J. Gen. Virol. 36:59-74 (1977); Hitt, M. M., et al.,“Structure and Genetic Organization of Adenovirus Vectors,” in TheDevelopment of Human Gene Therapy, Friedmann, T., ed., Cold SpringHarbor Laboratory Press, Cold Spring Harbor, N.Y. (1999), pp. 61-86;Kozarsky, K. F., and Wilson, J. M., Curr. Opin. Genet. Dev. 3:499-503(1993); Krougliak, V., and Graham, F. L., Hum. Gene Ther. 6:1575-1586(1995); Lochmuller, H., et al., Hum. Gene Ther. 5:1485-1491 (1994);Navarro, V., et al., Gene Ther. 6:1884-1892 (1999); Russell, W. C., J.Gen. Virol. 81:2573-2604 (2000); Wang, I. I., and Huang, I. I., DrugDiscovery Today 5:10-16 (2000); Wivel, N. A., “Adenoviral Vectors,” inThe Development of Human Gene Therapy, Friedmann, T., ed., Cold SpringHarbor Laboratory Press, Cold Spring Harbor, N.Y. (1999), pp. 87-110;and Zhang, W. W., et al., BioTechniques 18:444-447 (1995).

[0445] Viral infection is referred to in some procedures in thisexample, and viral transduction in other procedures. These terms aredefined below.

[0446] Infection: Applies to situations where viral replication occursand infectious viral progeny are generated. Only cell lines that stablyexpress E1 may be infected.

[0447] Transduction: Applies to situations where no viral replicationoccurs and no infectious viral progeny are generated. Mammalian celllines that do not express E1 are transduced. In this case, an adenovirusis used as a gene delivery vehicle.

[0448] The ViraPower™ Adenoviral Expression System is suitable for invivo gene delivery applications. Many groups have successfully usedadenoviral vectors to express a target gene in a multitude of tissuesincluding skeletal muscle, lung, heart, and brain. For more informationabout target genes that have been successfully expressed in vivo usingadenoviral-based vectors, refer to the publications, supra.

[0449] The ViraPower™ Adenoviral Expression System includes thefollowing safety features. The entire E1 region is deleted in thepAd/CMV/V5-DEST™ or pAd/PL-DEST™ expression vectors. Expression of theE1 proteins is required for the expression of the other viral genes(e.g., late genes), and thus viral replication only occurs in cells thatexpress E1. Adenovirus produced from the pAd/CMV/V5-DEST™ orpAd/PL-DEST™ expression vectors is replication-incompetent in anymammalian cells that do not express the E1a and E1b proteins. Adenovirusdoes not integrate into the host genome upon transduction. Because thevirus is replication-incompetent, the presence of the viral genome istransient and will eventually be diluted out as cell division occurs.For more information regarding adenoviral transduction and expression,see the publications listed supra.

[0450] Despite the presence of the safety features discussed above, theadenovirus produced with this system may still pose some biohazardousrisk since it can transduce primary human cells. For this reason,adenoviral stocks generated using this system be handled as BiosafetyLevel 2 (BL-2) organisms and strictly all published guidelines for BL-2should be followed. Furthermore, extra caution should be taken whencreating adenovirus carrying potential harmful or toxic genes (e.g.,activated oncogenes) or when producing large-scale preparations ofvirus. For more information about the BL-2 guidelines and adenovirushandling, refer to the document, “Biosafety in Microbiological andBiomedical Laboratories,” 4th Edition, published by the Centers forDisease Control (CDC). This document may be downloaded from the CDC Website.

[0451] The genomic copy of E1 in all 293 cell lines contains homologousregions of overlap with the pAd/CMV/V5-DEST™ and pAd/PL-DEST™ vectors.In rare instances, it is possible for homologous recombination to occurbetween the E1 genomic region in 293 cells and the viral DNA, causingthe gene of interest to be replaced with the E1 region, and resulting ingeneration of a “wild-type,” replication-competent adenovirus (RCA).This event is most likely to occur during large-scale preparation oramplification of virus, and the growth advantages of the RCA allow it toquickly overtake cultures of recombinant adenovirus. To reduce thelikelihood of propagating RCA-contaminated adenoviral stocks, cautionshould be used when handling all viral preparations, which is consideredto be BL-2 material. Routine screening for the presence of wild-type RCAcontamination after large-scale viral preparations should be performed.Suitable methods to screen for RCA contamination include PCR screeningor supernatant rescue assays. If RCA contamination occurs, plaquepurification may be performed to re-isolate the recombinant adenovirusof interest. As an alternative, E1-containing producer cell lines suchas 911 or PER.C6 which contain no regions of homologous overlap with theadenoviral vectors may be used to help reduce the incidence of RCAgeneration. For more information regarding RCA, see the publicationslisted supra, in particular Lochmuller, et al. (1994) and Zhang et al.(1995).

[0452]FIG. 13 describes the general steps required to express the geneof interest using the ViraPower™ Adenoviral Expression System. Forinstructions to generate an adenovirus expression clone usingpAd/CMV/V5-DEST™ or pAd/PL-DEST™, refer to the pAd/CMV/V5-DEST™ andpAd/PL-DEST™ GATEWAY™ Vector manual, available from InvitrogenCorporation, Carlsbad, Calif.

[0453] First, the adenovirus expression clone containing the gene ofinterest is generated and digested with Pac I to expose the ITRsaccording to the methods described herein or by published methods, e.g.,the pAd/PL-DEST™ and pAd/CMV/V5-DEST™ manuals, from InvitrogenCorporation, Carlsbad, Calif. Next, the 293A producer cell line istransfected with the adenovirus expression clone. The cells areharvested and lysed to produce a crude viral lysate. The adenovirus maybe amplified by infecting 293A producer cells with the crude virallysate, and the resulting viral stock is titered. The viral stock isused to infect a mammalian cell line of interest, which is then assayedfor expression of the gene of interest.

[0454] The ViraPower™ Adenoviral Expression System is designed to createan adenovirus to deliver and transiently express a gene of interest inmammalian cells. Although the system has been designed to express anyrecombinant polypeptide of interest in the simplest, most directfashion, use of the system is geared towards those users who arefamiliar with the biology of DNA viruses and adenoviral vectors andpossess a working knowledge of viral and tissue culture techniques. Formore information about these topics, refer to the following publishedreviews: Adenovirus biology: see Russell, W. C. J. Gen. Virol.81:2573-2604 (2000). Adenoviral vectors: see Hitt, M. M., et al.,“Structure and Genetic Organization of Adenovirus Vectors,” in TheDevelopment of Human Gene Therapy, Friedmann, T., ed., Cold SpringHarbor Laboratory Press, Cold Spring Harbor, N.Y. (1999), pp. 61-86, andWivel, N. A., “Adenoviral Vectors,” in The Development of Human GeneTherapy, Friedmann, T., ed., Cold Spring Harbor Laboratory Press, ColdSpring Harbor, N.Y. (1999), pp. 87-110. Adenovirus applications: seeWang, I. I., and Huang, I. I., Drug Discovery Today 5:10-16 (2000).

[0455] An expression clone may be created containing a DNA sequence ofinterest in pAd/CMV/V5-DEST™, which expresses the gene of interest underthe control of the human CMV promoter, or in pAd/PL-DEST™, which ispromoterless, thus allowing the insertion of a cassette containing thegene of interest under the control of any promoter. Refer to thepAd/CMV/V5-DEST™ and pAd/PL-DEST™ GATEWAY™ Vector manual for furtherinstructions. Once an expression clone has been created, any method ofpreparing purified plasmid DNA that is clean and free from phenol andsodium chloride may be used. Contaminants may kill the cells, and saltmay interfere with lipid complexing, decreasing transfection efficiency.Suitable methods of isolating plasmid DNA include, but are not limitedto, the S.N.A.P.™ MidiPrep Kit (Catalog No. K1910-01, InvitrogenCorporation, Carlsbad, Calif.) and cesium chloride gradientcentrifugation.

[0456] Any 293-derived cell line or other cell line that expresses theE1 proteins may be used to produce adenovirus. One such cell linesparticularly suited for use in the present invention is the human 293ACell Line, included with the ViraPower™ Adenoviral Expression kits tofacilitate adenovirus production from the E1-deleted pAd-DEST™ vectors.The 293A Cell Line, a subclone of the 293 cell line, supplies in transthe E1 proteins that are required for expression of adenoviral lategenes, and thus viral replication. The cell line exhibits a flattenedmorphology, enabling easier visualization of plaques. For moreinformation about how to culture and maintain 293A cells, refer to the293A Cell Line manual, available from Invitrogen Corporation, Carlsbad,Calif.

[0457] Once an expression clone, for example a pAd-DEST™ expressionclone, is created, the expression clone is transfected into a suitablehost cell line (e.g., 293A cells) to produce an adenoviral stock. Thefollowing section provides protocols and instructions to generate anadenoviral stock, using pAd-DEST™ to illustrate the method of thepresent invention.

[0458] Before transfecting a pAd-DEST™ expression clone into 293A cells,the left and right viral ITRs are exposed to allow proper viralreplication and packaging. Each pAd-DEST™ vector contains Pac Irestriction sites (refer to the maps of each vector in thepAd/CMV/V5-DEST™ and pAd/PL-DEST™ manual for the location of the Pac Isites). Digestion of the vector with Pac I allows exposure of the leftand right viral ITRs and removal of the bacterial sequences (i.e., pUCorigin and ampicillin resistance gene). The DNA sequence of interestshould not contain any Pac I restriction sites. At least 5 mg ofpurified plasmid DNA of the pAd-DEST™ expression construct is digestedwith Pac I (New England Biolabs, Catalog No. R0547S) according to themanufacturer's instructions. The digested plasmid DNA may be purifiedusing phenol/chloroform extraction followed by ethanol precipitation ora DNA purification kit (e.g., Invitrogen's S.N.A.P. MiniPrep™ Kit;catalog No. K1900-01). Gel purification is not required. The purifiedplasmid is resuspended or eluted, as appropriate, in sterile water or TEBuffer, pH 8.0 to a final concentration of 0.1-3.0 mg/ml.

[0459] The following materials are required before beginning: PacI-digested pAd-DEST™ expression clone containing the DNA sequence ofinterest (0.1-3.0 mg/ml in sterile water or TE, pH 8.0);pAd/CMV/V5-GW/lacZ positive control vector (supplied with the kit;resuspended in sterile water to a concentration of 1 mg/ml); 293A cellscultured in the appropriate medium (see the 293A Cell Line manual fordetails); transfection reagent suitable for transfecting 293A cells(e.g., Lipofectamine™ 2000); Opti-MEM® I Reduced Serum Medium (if usingLipofectamine™ 2000; pre-warmed); fetal bovine serum (FBS); sterile6-well and 10 cm tissue culture plates; and sterile tissue culturesupplies, e.g., 15 ml sterile, capped, conical tubes, table-topcentrifuge, water bath (set to 37° C.), and cryovials.

[0460] The pAd/CMV/V5-GW/lacZ plasmid is included with each ViraPower™Adenoviral Expression kit as a positive control vector for expression.The positive control vector may be included in the transfectionexperiment to generate a control adenoviral stock that may be used tohelp optimize expression conditions in the mammalian cell line ofinterest. For more information about the positive control vector, referto the pAd/CMV/V5-DEST™ and pAd/PL-DEST™ GATEWAY™ Vector manual.

[0461] Any suitable transfection reagent may be used to introduce thepAd-DEST™ expression construct into 293A cells. Particularly suitable isthe cationic lipid-based Lipofectamine™ 2000 Reagent available fromInvitrogen. Using Lipofectamine™ 2000 to transfect 293A cells offersseveral advantages: provides the highest transfection efficiency in 293Acells; DNA-Lipofectamine™ 2000 complexes can be added directly to cellsin culture medium in the presence of serum; and removal of complexes ormedium change or addition following transfection are not required,although complexes can be removed after 4-6 hours without loss ofactivity. To facilitate optimal formation of DNA-Lipofectamine™ 2000complexes, the Opti-MEM® I Reduced Serum Medium available fromInvitrogen may be used. For more information about Opti-MEM® I, contactInvitrogen Corporation, Carlsbad, Calif.

[0462] Provided below is one method by which adenoviral stocks may beproduced in 293A cells using the following optimized transfectionconditions below. The amount of adenovirus produced using theserecommended conditions is approximately 10 ml of crude viral lysate witha titer ranging from 1×10⁷ to 1×10⁸ plaque-forming units (pfu)/ml.Lipofectamine™ 2000 is one suitable transfection reagent. Othertransfection reagents are readily available and may be used according tothe appropriate protocols. Condition Amount Tissue culture plate size6-well (one well per adenoviral construct) Number of 293 A cells totransfect 5 × 10⁵ cells (see Note below) Amount of Pac I-digestedpAd-DEST ™ 1 μg expression plasmid Amount of Lipofectamine ™ 2000 3 μl

[0463] 293A cells are plated 24 hours prior to transfection in completemedium, and should be healthy and 90-95% confluent on the day oftransfection.

[0464] Provided herein is a method to transfect 293A cells usingLipofectamine™ 2000. One feature of the provided method is that cellsmay be kept in culture medium during transfection. A positive controland a negative control (no DNA, no Lipofectamine™ 2000) may be includedthe experiment to aid in evaluation of the results.

[0465] The day before transfection, the 293A cells are trypsinized andcounted, then plated at 5×10⁵ cells per well in a 6-well platecontaining 2 ml of normal growth medium containing serum. On the day oftransfection, the culture medium from the 293A cells is removed andreplaced with 1.5 ml of normal growth medium containing serum (orOpti-MEM® I Medium containing serum). Antibiotics should not included.

[0466] The DNA-Lipofectamine™ 2000 complexes are prepared for eachtransfection sample as follows: 1 μg of Pac I-digested pAd-DEST™expression plasmid DNA is diluted in 250 μl of Opti-MEM® I Mediumwithout serum and mixed gently. The Lipofectamine™ 2000 reagent is mixedgently before use, then diluted 3 μl in 250 μl of Opti-MEM® I Mediumwithout serum. The solution is mixed gently and incubated for 5 minutesat room temperature. After the 5 minute incubation, the diluted DNA iscombined with the diluted Lipofectamine™ 2000 and mixed gently. Thesolution is then incubated for 20 minutes at room temperature to allowthe DNA-Lipofectamine™ 2000 complexes to form. The solution may appearcloudy, but this will not impede the transfection. TheDNA-Lipofectamine™ 2000 complexes is added dropwise to each well andmixed gently by rocking the plate back and forth. The cells areincubated overnight at 37° C. in a CO₂ incubator.

[0467] The next day, the medium containing the DNA-Lipofectamine™ 2000complexes is removed and replaced with complete culture medium (i.e.,D-MEM containing 10% FBS, 2 mM L-glutamine, and 1%penicillin/streptomycin). 48 hours post transfection, the cells aretrypsinized and transferred to a sterile 10 cm tissue culture platecontaining 10 ml of complete culture medium. The recommended guidelinesfor working with BL-2 organisms should be followed throughout theseprocedures. The culture medium is replaced with fresh, complete culturemedium every 2-3 days until visible regions of cytopathic effect (CPE)are observed (typically 7-10 days post-transfection). The infectionsproceed until approximately 80% CPE is observed (typically 10-13 dayspost-transfection). The recombinant adenovirus-containing cells areharvested by squirting cells off the plate with a 10 ml tissue culturepipette. The cells and media are transferred to a sterile, 15 ml, cappedtube for lysing as described below.

[0468] In this example, Pac I-digested pAd/CMV/V5-GW/lacZ plasmid wastransfected into 293A cells using the protocol described supra. FIGS.14A-C show transfected cells as they undergo CPE.

[0469] Day 4-6 post-transfection (FIG. 14A): at this early stage, cellsproducing adenovirus first appear as patches of rounding, dying cells.

[0470] Day 6-8 post-transfection (FIG. 14B): as the infection proceeds,cells containing viral particles lyse and infect neighboring cells. Aplaque begins to form.

[0471] Day 8-10 post-transfection (FIG. 14C): at this late stage,infected neighboring cells lyse, forming a plaque that is clearlyvisible.

[0472] After the adenovirus-containing cells and media are harvested,several freeze/thaw cycles followed by centrifugation may be used toprepare a crude viral lysate. The freeze/thaw cycles cause the cells tolyse and allow release of intracellular viral particles. The tubecontaining harvested transfected cells and media is placed at −80° C.for 30 minute, then placed in a 37° C. water bath for 15 minutes tothaw. The freezing and thawing steps are repeated twice. The cell lysateis centrifuged in a table-top centrifuge at 3000 rpm for 15 minutes atroom temperature to pellet the cell debris. The supernatant containingviral particles, the viral stock, may be transferred to cryovials in 1ml aliquots and stored at −80° C.

[0473] Once a crude viral stock is prepared, it may be amplified byinfecting 293A cells as described below. This procedure is recommendedto obtain the highest viral titers and optimal results in transductionstudies. The titer of the crude viral stock may be determined, and thisstock may be used to transduce the mammalian cells of interest to verifythe functionality of the adenoviral construct in preliminary expressionexperiments.

[0474] The viral stocks are placed at −80° C. for long-term storage.Because adenovirus is non-enveloped, viral stocks remain relativelystable and some freezing and thawing of the viral stocks is acceptable.Freezing and thawing viral stocks more than 10 times should be avoidedas loss of viral titer can occur. When stored properly, viral stocks ofan appropriate titer should be suitable for use for up to one year.After long-term storage, re-titering the viral stocks may be performedbefore use.

[0475] Once a crude viral stock is created, this stock may be used toinfect 293A cells to generate a higher titer viral stock (i.e., amplifythe virus). The titer of the initial viral stock obtained fromtransfecting 293A cells generally ranges from 1×10⁷ to 1×10⁸plaque-forming units (pfu)/ml. Amplification allows production of aviral stock with a titer ranging from 1×10⁸ to 1×10⁹ pfu/ml and isgenerally recommended. Guidelines and protocols are provided in thisexample to amplify the recombinant adenovirus using 293A cells plated ina 10 cm dish. Larger-scale amplification is possible. Other 293 celllines or cell lines expressing the E1 proteins are also suitable.

[0476] The recommended Federal guidelines for working with BL-2organisms should be followed for all work with infectious virus. Allmanipulations should be performed within a certified biosafety cabinet.Media containing virus should be treated with bleach. Used pipettes,pipette tips, and other tissue culture supplies should be treated withbleach or disposed of as biohazardous waste. Gloves, a laboratory coat,and safety glasses or goggles should be worn when handling viral stocksand media containing virus.

[0477] Wild-type RCA contamination has not been observed in small-scale(i.e., 3×10⁶ 293A cells plated in a 10 cm dish) adenoviral amplificationusing the protocol provided below. However, large-scale amplification ofvirus should be screened for wild-type RCA contamination. Even inlarge-scale preparations, contamination of adenoviral stocks withwild-type RCA is a rare event.

[0478] The following materials are required for amplifying the viralstock: crude adenoviral stock of the pAd-DEST™ construct; sterile 10 cmtissue culture plates; sterile, tissue culture supplies 15 ml sterile,capped, conical tubes; equipment and supplies such as table-topcentrifuge, 37° C. water bath, and cryovials.

[0479] A typical infection of 293A cells uses the following conditions:Condition Amount Tissue culture plate size 10 cm (one per adenoviralconstruct) Number of 293 A cells to infect 3 × 10⁶ cells Amount of crudeadenoviral stock 100 μl to use

[0480] For infection, a 10 cm plate of 293A cells is infected with 100μl of untitered crude viral stock. Assuming a viral titer of 1×10⁷ to1×10⁸ pfu/ml, this generally allows harvesting the desired numberadenovirus-containing cells 2-3 days after infection. The volume ofcrude viral stock used to infect cells, may be varied proportionallyaccording to the desired number of cells and/or amount of crude viralstock to as much as 1 ml of crude viral stock. If the titer of the crudeviral stock is known, 293A cells are infected at a multiplicity ofinfection (MOI)=3 to 5.

[0481] The procedure below may be used to amplify the adenoviral stockusing 293A cells. The day before infection, the 293A cells aretrypsinized and counted before plating them at 3×10⁶ cells per 10 cmplate. Cells are plated in 10 ml of normal growth medium containingserum. On the day of infection, the cells are verified to be at 80-90%confluency before proceeding. The desired amount of crude adenoviralstock (e.g., 100 μl) is added to the cells. The plate is swirled gentlyto mix. The cells are incubated at 37° C. in a CO₂ incubator and theinfection is allowed to proceed until 80-90% of the cells have roundedup and are floating or lightly attached to the tissue culture dish(typically 2-3 days post-infection). This CPE indicates that cells areloaded with adenoviral particles. Using less than 100 μl of crude viralstock or a lower titer stock for infection, may require a longerincubation to achieve CPE. The adenovirus-containing cells are harvestedby squirting cells off the plate with a 10 ml tissue culture pipette.The cells and media are transferred to a sterile, 15 ml, capped tubewhich is then placed at −80° C. for 30 minutes. The tube is removed andplaced in a 37° C. water bath for 15 minutes to thaw. The freezing andthawing steps are repeated twice. The cell lysate is centrifuged in atable-top centrifuge at 3000 rpm for 15 minutes at room temperature topellet the cell debris. The supernatant containing viral particles istransferred to cryovials in 1 ml aliquots and may be stored at −80° C.

[0482] The amplification procedure is easily scalable to any size tissueculture dish or roller bottle. If it is desirable to scale up theamplification, the number of cells and amount of crude viral stock andmedium used is increased in proportion to the difference in surface areaof the culture vessel. A screen for the presence of wild-type RCAcontamination in the amplified stock may be performed according tosuitable screening protocols as described in published literature knownto those skilled in the art.

[0483] Before proceeding to transduce the mammalian cell line ofinterest and express the polynucleotide of interest or recombinantpolypeptide, determining the titer of the adenoviral stock may beuseful. While this procedure is not required for some applications, itis necessary if the number of adenoviral particles introduced to eachcell is to be controlled and to generate reproducible expressionresults. Guidelines and protocols are provided in this example.

[0484] To determine the titer of an adenoviral stock, 293A cells areplated in 6-well tissue culture plates. Ten-fold serial dilutions of theadenoviral stock are prepared, then used to infect 293A cells overnight.A plaque assay is performed by first overlaying the infected 293A cellswith an agarose/plaquing media solution then allowing 8-12 days forplaques to form. The cells are stained and the number of plaques arecounted in each dilution

[0485] A number of factors may influence viral titers. Titers generallydecrease as the size of the insert increases. The size of the wild-typeadenovirus type 5 genome is approximately 35.9 kb. Studies havedemonstrated that recombinant adenovirus can efficiently package up to108% of the wild-type virus size from E1 and E3-deleted vectors. Takinginto account the size of the elements required for expression from eachpAd-DEST™ vector, the DNA sequence or gene of interest should not exceedthe size indicated below for efficient packaging. Vector Insert SizeLimit pAd/CMV/V5-DEST ™ 6.0 kbp Ad/PL-DEST ™ 7.5 kb

[0486] Other factors include the characteristics of the cell line usedfor titering and the age of the adenoviral stock. Viral titers maydecrease with long-term storage at −80° C. If the adenoviral stock hasbeen stored for 6 months to 1 year, re-titering the adenoviral stock maybe performed prior to use in an expression experiment. The number offreeze/thaw cycles and storage of the adenoviral stock may also affecttiter. A limited number of freeze/thaw cycles is acceptable, but viraltiters may decrease with more than 10 freeze/thaw cycles. Adenoviralstocks may be aliquotted and stored at −80° C.

[0487] The 293A cell line supplied with the kit is particularly suitablefor use in titering the adenoviral stock, however other cell lines maybe used. If another cell line is used, it should: express the E1proteins, grow as an adherent cell line, be easy to handle, exhibit adoubling time in the range of 18-25 hours, and be non-migratory.

[0488] The titer of an adenoviral construct may vary depending on whichcell line is chosen. If more than one adenoviral construct is betitered, all of the adenoviral constructs is preferably titered usingthe same mammalian cell line.

[0489] To determine the titer of the adenoviral construct, the followingmaterials are required: the pAd-DEST™ adenoviral stock (stored at −80°C. until use); 293A Cell Line or other appropriate mammalian cell lineof choice (see above); complete culture medium for the cell line; 6-welltissue culture plates; 4% agarose (see Recipes; equilibrated to 65° C.before use); plaquing media (normal growth medium containing 2% FBS;equilibrated to 37° C. before use); and 5 mg/ml MTT solution or otherappropriate reagent for staining (see Recipes; see below foralternatives).

[0490] The vital dye,3-[4,5-Dimethylthiazol-2-yl]-2,5-diphenyltetrazolium bromide (thiazolylblue (MTT)) is suitable for use as a staining reagent to help visualizeplaques. Other vital stains including Neutral Red (Sigma-Aldrich, St.Louis, Mo., catalog No. N7005) are suitable. To use Neutral Red, a 1%solution (100× stock solution) is prepared in water and stored at +4° C.

[0491] The procedure presented herein is a method to determine the titerof the adenoviral stock using the 293A cell line or other appropriatecell line. Other suitable methods are available and known in the art. Atleast one 6-well plate is required for every adenoviral stock to betitered (six dilutions or one mock well and five dilutions). If anadenoviral stock of the pAd/CMV/V5-GW/lacZ positive expression controlhas been generated, titering this stock may be done as well. The daybefore infection (Day 1), the cells are trypsinized and counted forplating at a density such that they will be 80-90% confluent at the timeof infection. For example, 293A cells may be used to titer theadenoviral stock and 1×10⁶ cells per well may be plated in each well ofa 6-well plate. The cells are incubated at 37° C. overnight.

[0492] On the day of infection (Day 2), the adenoviral stock is thawedand diluted 10-fold serially to concentrations ranging from 10⁻⁴ to10⁻⁹. For each dilution, the adenoviral construct is diluted intocomplete culture medium to a final volume of 1 ml and mixed by gentleinversion. The culture medium is removed from the cells, and thedilutions are added to one well of cells (total volume=1 ml). The plateis swirled gently to disperse the media, then incubated at 37° C.overnight. The following day (Day 3), the media containing virus isremoved and the cells are gently overlaid with 2 ml of Agarose Overlaysolution per well.

[0493] An agarose overlay solution (enough to overlay one 6-well plateat a time) may be prepared as follows. For one 6-well plate (2 mloverlay per well), 12 ml of pre-warmed (at 37° C.) Plaquing Media and1.2 ml of pre-warmed (at 65° C.) 4% Agarose is gently mixed whileavoiding the formation of bubbles. The overlay is applied to the cellsby gently pipetting the overlay down the side of each aspirated wellwhile working quickly to prevent premature solidification. The 6-wellplate is placed in a level tissue-culture hood at room temperature for15 minutes or until the Agarose Overlay solidifies. The plate isreturned to a 37° C. humidified CO₂ incubator. 3-4 days following theinitial overlay (Day 6-7), the cells are gently overlaid with anadditional 1 ml of Agarose Overlay solution (prepared as before) perwell. The Agarose Overlay is allowed to solidify before returning theplate to a 37° C. humidified CO₂ incubator. The plates are monitoreduntil plaques are visible (generally 8-12 days post-infection). For eachwell, the 5 mg/ml MTT solution (1/10 the volume of the Agarose Overlay)is layered gently on top of the solidified agar to stain. For example,if each well contains 3 ml of Agarose Overlay, 300 μl of 5 mg/ml MTT isused. The plates are incubated for 3 hours at 37° C. The plaques arecounted to determine the titer of the adenoviral stock.

[0494] When titering pAd/CMV/V5-DEST™ or pAd/PL-DEST™ adenoviral stocksusing 293A cells, titers ranging from 1×10⁸ to 1×10⁹ pfu/ml areobtained. Adenoviral constructs with titers in this range are generallysuitable for use in most applications. If the titer of the adenoviralstock is less than 1×10⁷ pfu/ml, a new adenoviral stock may be producedto increase the titer. See the Troubleshooting section below for moretips and guidelines to optimize the viral yield.

[0495] For some applications, viral titers higher than 1×10⁹ pfu/ml maybe desired. It is possible to concentrate adenoviral stocks using avariety of methods (e.g., CsCl purification; Engelhardt, J.F., et al.,Nature Genetics 4:27-34 (1993), without significantly affecting theirtransducibility. Use of these methods allows generation of adenoviralstocks with titers as high as 1×10¹¹ pfu/ml.

[0496] Once an adenoviral stock with a suitable titer is generated, itmay be used to transduce the adenoviral construct into the mammaliancell line of choice and assay for expression of the polynucleotide ofinterest. Guidelines illustrating one method of transduction areprovided below, though it will be appreciated that many such methods areknown in the art and may be used in the present invention.

[0497] The pAd/CMV/V5-DEST™ or pAd/PL-DEST™ adenoviral construct isreplication-incompetent and does not integrate into the host genome.Therefore, once transduced into the mammalian cells of choice, the geneof interest will be expressed only as long as the viral genome ispresent. The adenovirus terminal protein (TP) covalently binds to theends of the viral DNA, and helps to stabilize the viral genome in thenucleus. In actively dividing cells, the adenovirus genome is graduallydiluted out as cell division occurs, resulting in an overall decrease intransgene expression over time (generally to background levels within1-2 weeks after transduction). In non-dividing cells (e.g., quiescentCD34+ cells) or animal tissues (e.g., skeletal muscle, neurons),transgene expression is more stable and can persist for as long as 6months following transduction.

[0498] In actively dividing cells (i.e., doubling time of approximately24 hours), transgene expression is generally detectable within 24 hoursof transduction, with maximal expression observed at 48-96 hours (2-4days) post transduction. Expression levels generally start to decline by5 days after transduction. In cell lines that exhibit longer doublingtimes or non-dividing cell lines, high levels of transgene expressiontypically persist for a longer time. If transducing the adenoviralconstruct into the mammalian cell line for the first time, a time courseof expression may be performed to determine the optimal conditions forexpression of the gene of interest.

[0499] To obtain optimal expression of the gene of interest, theadenoviral construct may be transduced into the mammalian cell line ofchoice using a suitable MOI. MOI is defined as the number of virusparticles per cell and generally correlates with expression. Typically,expression levels increase linearly as the MOI increases.

[0500] A number of factors can influence determination of an optimal MOIincluding the nature of the mammalian cell line to be used (e.g.,non-dividing vs. dividing cell type), its transduction efficiency, theapplication of interest, and the nature of the gene of interest. Iftransducing the adenoviral construct into the mammalian cell line ofchoice for the first time, using a range of MOIs (e.g., 0, 0.5, 1, 2, 5,10, 20, 50) to determine the MOI required to obtain optimal expressionof the DNA or interest or recombinant polypeptide may be performed.

[0501] In general, 80-90% of the cells in an actively dividing cell line(e.g., HT1080) express a target gene when transduced at an MOI of ˜1.Other cell types including non-dividing cells may transduce adenoviralconstructs less efficiently. If transducing the adenoviral constructinto a non-dividing cell type, the MOI may be increased to achieveoptimal expression levels for the polynucleotide of interest orrecombinant polypeptide.

[0502] The pAd/CMV/V5-GW/lacZ control adenoviral construct may be usedto determine the optimal MOI for the particular cell line andapplication. Once the Ad/CMV/V5-GW/lacZ adenovirus is transduced intothe mammalian cell line of choice, the gene encoding β-galactosidasewill be constitutively expressed and can be easily assayed (refer to thepAd/CMV/V5-DEST™ and pAd/PL-DEST™ GATEWAY™ Vector manual for details,available from Invitrogen Corporation, Carlsbad, Calif.).

[0503] Viral supernatants are generated by lysing cells containing virusinto spent media harvested from the 293A producer cells. Spent medialacks nutrients and may contain some toxic waste products. If a largevolume of viral supernatant is used to transduce the mammalian cell line(e.g., 1 ml of viral supernatant per well in a 6-well plate), growthcharacteristics or morphology of the target cells may be affected duringtransduction. These effects are generally alleviated after transductionwhen the media is replaced with fresh, complete media.

[0504] The procedure described herein illustrates one method totransduce the mammalian cell line of choice with the adenoviralconstruct. Other methods suitable for use with the present invention arereadily available for use by one skilled in the art. Mammalian cells ofchoice are plated in complete media. On the day of transduction (Day 1),the adenoviral stock is thawed, and the appropriate amount of virus isdiluted (if necessary) into fresh complete medium. The culture medium isremoved from the cells. The medium containing virus is mixed gently bypipetting and add to the cells. The plate is swirled gently to dispersethe medium, then incubated at 37° C. overnight. On the following day(Day 2), the medium containing virus is removed and replaced with fresh,complete culture medium. The cells are harvested (if needed) on thedesired day (e.g., 2 days post transduction) and assayed for expressionof the polynucleotide of interest or recombinant polypeptide.

[0505] Any method of choice to detect the polynucleotide of interest orrecombinant polypeptide of interest including functional analysis,immunofluorescence, northern blot, or western blot. If the gene ofinterest is cloned in frame with an epitope tag, the recombinantpolypeptide of interest may be detected using an antibody to the epitopetag (see the pAd/CMV/V5-DEST™ and pAd/PL-DEST™ GATEWAY™ Vector manualfor details, available from Invitrogen, Carlsbad, Calif.).

[0506] Troubleshooting

[0507] Below are listed some potential problems and possible solutionsthat may help troubleshoot the cotransfection and titering experiments.Problem Reason Solution Low viral Low transfection efficiency: Repeatthe Pac I digestion. Make titer Incomplete Pac I digestion or sure thatthe purified DNA is not digested DNA contaminated contaminated withphenol, ethanol, or with phenol, ethanol, or salts salts. Use healthy293A cells; do not Unhealthy 293A cells; cells overgrow. Cells should be90-95% exhibit low viability 293A confluent at the time of transfection.cells plated too sparsely Optimize such that plasmid DNA (in PlasmidDNA:transfection μg): Lipofectamine ™ 2000 (in μl) reagent ratioincorrect ratio ranges from 1:2 to 1:3. If using another transfectionreagent, optimize according to the manufacturer's recommendations. Viralsupernatant too dilute Concentrate virus using CsCl purification(Engelhardt, J. F., et al., Nature Genetics 4: 27-34 (1993) or anymethod of choice. Viral supernatant frozen and Do not freeze/thaw viralsupernatant thawed multiple times more than 10 times. Gene of interestis large Viral titers generally decrease as the size of the insertincreases; inserts larger than 6 kb (for pAd/CMV/V5-DEST ™) and 7.5 kb(for pAd/PL-DEST ™) are not recommended. Gene of interest is toxic toGeneration of constructs containing cells activated oncogenes orpotentially harmful genes is not recommended. No plaques Viral stocksstored Aliquot and store stocks at −80° C. obtained incorrectly Do notfreeze/thaw more than 10 times. upon titering Incorrect titering cellline Use the 293A cell line or any cell line used with thecharacteristics discussed. Agarose overlay incorrectly Make sure thatthe agarose is not too prepared hot before addition to the cells; hotagarose will kill the cells.

[0508] Transducing Mammalian Cells

[0509] Below are listed some potential problems and possible solutionsthat may help troubleshoot the transduction and expression experiment.Problem Reason Solution Titer Viral supernatant not diluted Titeradenovirus using 10-fold serial indeterminable; sufficiently dilutionsranging from 10⁻⁴ to 10⁻⁹. cells confluent No expression Viral stocksstored Aliquot and store stocks at −80° C. incorrectly Do notfreeze/thaw more than 10 times. Gene of interest contains a Performmutagenesis to change or Pac I site remove the Pac I site. Poorexpression Poor transduction efficiency: Mammalian cells not healthyMake sure that the cells are healthy Non-dividing cell type used beforetransduction. Transduce the adenoviral construct into cells using ahigher MOI. MOI too low Transduce the adenoviral construct into cellsusing a higher MOI. Low viral titer Amplify the adenoviral stock usingthe procedure. Adenoviral stock Screen for RCA contaminationcontaminated with RCA (Dion, L. D., et al., J. Virol. Methods 56: 99-107(1996)). Prepare a new adenoviral stock or plaque purify to isolaterecombinant adenovirus. Cells harvested too soon Do not harvest cellsuntil at least after transduction 24-48 hours after transduction. Cellsharvested too long For actively dividing cells, assay for aftertransduction maximal levels of recombinant polypeptide expression within5 days of transduction. Gene of interest is toxic to Generation ofconstructs containing cells activated oncogenes or potentially harmfulgenes is not recommended. Persistent Too much crude viral stock Reducethe amount crude viral stock toxicity in used used for transduction ordilute the target cells crude viral stock. Amplify the adenoviral stock.Concentrate the crude viral stock. Wild-type RCA Screen for RCAcontamination contamination (Dion, L. D., et al., J. Virol. Methods 56:99-107 (1996); Zhang, W. W., et al., BioTechniques 18: 444-447 (1995).Plaque purify to isolate recombinant adenovirus or prepare a newadenoviral stock.

Recipes

[0510] 4% Agarose

[0511] This procedure may be used to prepare a 4% Agarose solution.

[0512] Materials Needed: Ultra Pure Agarose (Invitrogen, Catalog No.15510-027) Deionized, sterile water.

[0513] Protocol: Prepare a 4% stock solution in deionized, sterilewater.

[0514] Autoclave at 121° C. for 20 minutes to sterilize. Equilibrate to65° C. in a water bath and use immediately or store at room temperatureindefinitely. If the agarose solution is stored at room temperature,melting the agarose is required before use. To melt, microwave theagarose to melt, then equilibrate to 65° C. in a water bath before use.

[0515] 5 mg/ml MTT

[0516] This procedure may be used to prepare a 5 mg/ml MTT solution.

[0517] Materials Needed:3-[4,5-Dimethylthiazol-2-yl]-2,5-diphenyl-tetrazolium bromide; Thiazolylblue (MTT; Sigma-Aldrich, St. Louis, Mo., Catalog No. M2128).Phosphate-Buffered Saline (PBS; Invitrogen, Catalog No. 10010-023).

[0518] Protocol: Prepare a 5 mg/ml stock solution in PBS.Filter-sterilize and dispense 5 ml aliquots into sterile, conical tubes.Store at +4° C. for up to 6 months.

Example 5

[0519] The present invention provides materials and methods for thestable expression of heterologous polypeptides in cells (e.g., insectcells). pIB/V5-His-DEST and pIB/V5-His-GW/lacZ are nucleic acidmolecules of the invention that are commercially available fromInvitrogen Corporation, Carlsbad, Calif. Information concerning theconstruction and use of these vectors may be found in Catalog no.12550-018 Version A, Jul. 15, 2002, 25-0607, available from InvitrogenCorporation, Carlsbad, Calif.

[0520] Nucleic acid molecules of the invention may be used to express apolypeptide of interest as part of a fusion polypeptide. Numeroussuitable fusion partners are known to those in the art. For example apolypeptide of interest may be expressed as a fusion polypeptidecontaining the V5 epitope. Antibodies to detect the V5 epitope, a 14amino acid epitope derived from the P and V proteins of theparamyxovirus, SV5 having the sequence GKPIPNPLLGLDST (Southern, J. A.,et al., J. Gen. Virol. 72:1551-1557 (1991)) are commercially availablefrom Invitrogen Corporation, Carlsbad, Calif., for example, Anti-V5Antibody catalog no. R960-25, Anti-V5-HRP Antibody catalog no. R961-25,and catalog no. Anti-V5-AP Antibody R962-25. A polypeptide of interestmay be expressed as a fusion polypeptide with a polyhistidine sequence.Antibodies to detect a polyhistidine sequence are commercially availablefrom Invitrogen Corporation, Carlsbad, Calif. For example,Anti-His(C-term) Antibody catalog no. R930-25, Anti-His(C-term)-HRPAntibody catalog no. R931-25, and Anti-His(C-term)-AP Antibody R932-25,all of which detect a C-terminal polyhistidine (6×His) tag and requirethe free carboxyl group for detection (i.e., detect the sequenceHHHHHH-COOH, see Lindner, P., et al., BioTechniques 22:140-149 (1997)).

[0521] An open reading frame present on a sequence of interest may becloned in frame with the C-terminal peptide containing the V5 epitopeand the polyhistidine (6×His) and Immobilized Metal AffinityChromatography (IMAC) may be used to purify the recombinant fusionpolypeptide. The ProBond™ Purification System as well as the Ni-NTAPurification System are available from Invitrogen Corporation, Carlsbad,Calif. Product Catalog no. ProBond ™ Purification System K850-01ProBond ™ Nickel-chelating Resin R801-01 R801-15 ProBond ™ PurificationSystem with K853-01 Anti-His(C-term)-HRP Antibody ProBond ™ PurificationSystem with K854-01 Anti-V5-HRP Antibody Purification Columns (10 mlR640-50 polypropylene columns) Ni-NTA Purification System K950-01 Ni-NTAAgarose R901-01 R901-15 Ni-NTA Purification System with K953-01Anti-His(C-term)-HRP Antibody Ni-NTA Purification System with K954-01Anti-V5-HRP Antibody

[0522] pIB/V5-His-DEST is a 5.0 kb vector derived from pIB/V5-His andadapted for use with GATEWAY™ Technology. It is designed to allowtransient or stable expression of a sequence of interest, which mayencode a polypeptide, in insect cell lines.

[0523] pIBNV5-His-DEST contains the following features: Feature BenefitOpIE2 promoter Allows constitutive expression of the gene of interest inlepidopteran insect cells (Theilmann, D. A., and Stewart, S., Virology187: 84-96 (1992)) attR1 and attR2 sites Allows recombinational cloningof the gene of interest from an entry clone. Chloramphenicol Allowscounterselection of resistance gene (Cm^(R)) expression clones. ccdBgene Allows negative selection of expression clones. V5 epitope Allowsdetection of a recombinant polypeptide with the Anti-V5 Antibodies(Southern, J. A., et al., J. Gen. Virol. 72: 1551-1557 (1991))C-terminal poly- Allows purification of recombinant histidine tagpolypeptides on metal-chelating resin such as ProBond ™ or Ni-NTA.Allows detection of the recombinant polypeptide by the Anti-His (C-term)Antibodies (Lindner, P., et al., BioTechniques 22: 140-149 (1997)) OpIE2polyadenylation Efficient transcription termination sequence andpolyadenylation of mRNA (Theilmann, D. A., and Stewart, S., Virology187: 84-96 (1992)) pUC origin Allows high-copy number replication andgrowth in E. coli. GP64 promoter Allows constitutive expression of theblasticidin resistance gene in lepidopteran insect cells (Blissard, G.W., et al., Virology 190: 783-793 (1992); Blissard, G. W., and Rohrmann,G. F., J. Virology 65: 5820-5827 (1991)) EM7 promoter Allows efficientexpression of the blasticidin and ampicillin resistance genes in E.coli. Blasticidin Allows generation of stable insect resistance gene(bsd) cell lines (Kimura, M., et al., Biochim. Biophys. ACTA 1219:653-659 (1994)) Ampicillin resistance Allows selection of transformantsgene (bla) in E. coli Note: The native promoter has been removed.Transcription is assumed to start from the EM7 promoter.

[0524] A map of pIB/V5-His-DEST is provided in FIG. 15 and thenucleotide sequence of the vector is provided in Table 12.

[0525] GATEWAY™ is a universal cloning technology that takes advantageof the site-specific recombination properties of bacteriophage lambda(Landy, 1989) to provide a rapid and highly efficient way to move a geneof interest into multiple vector systems. To express a sequence ofinterest using GATEWAY™ Technology: clone the sequence of interest intoa GATEWAY™ entry vector to create an entry clone; generate an expressionclone by performing an LR recombination reaction between the entry cloneand a GATEWAY™ destination vector (e.g. pIB/V5-His-DEST); and introducethe expression clone into insect cells for transient or stableexpression.

[0526] Baculovirus immediate-early promoters utilize the host celltranscription machinery and do not require viral factors for activation.The OpIE2 promoter is from the baculovirus Orgyia pseudotsugatamulticapsid nuclear polyhedrosis virus (OpMNPV) and drives constitutiveexpression of the gene of interest in pIB/V5-His-DEST. The virus'natural host is the Douglas fir tussock moth; however, the promoterallows protein expression in Lymantria dispar (LD652Y), Spodopterafrugiperda cells (Sf9) (Hegedus, D. D., et al., Gene 207:241-249 (1998);Pfeifer, T. A., et al., Gene 188:183-190 (1997)), Sf21 (Invitrogen),Trichoplusia ni (High Five™, Invitrogen Corporation, Carlsbad, Calif.),Drosophila (Kc1, S2) (Hegedus, D. D., et al., Gene 207:241-249 (1998);Pfeifer, T. A., et al., Gene 188:183-190 (1997)) and mosquito celllines. The OpIE2 promoter has been sequenced and analyzed. The sequenceof the promoter is provided in FIG. 16.

[0527] Although the OpIE2 promoter provides relatively high levels ofconstitutive expression, some proteins may not be expressed at levelsseen with baculovirus late promoters such as polyhedrin or very latepromoters such as p10 (Jarvis, D. L., et al., Protein Expression andPurification 8:191-203 (1996)). Typical expression levels range from 1-2μg/ml (human IL-6; Invitrogen) to 8-10 μg/ml (human melanotransferrin)(Hegedus, D. D., et al., Protein Expression and Purification 15:296-307(1999)).

[0528] The OpIE2 promoter has been analyzed by deletion analysis using aCAT reporter in both Lymantria dispar (LD652Y) and Spodoptera frugiperda(Sf9) cells. Expression in Sf9 cells was much higher than in LD652Ycells. Deletion analysis revealed that sequence up to −275 base pairsfrom the start of transcription is necessary for maximal expression(Theilmann, D. A., and Stewart, S., Virology 187:84-96 (1992)).Additional sequence beyond −275 may broaden the host range expression ofthis plasmid to other insect cell lines In addition, an 18 bp elementappears to be required for expression. This 18 bp element is repeatedalmost completely in three different locations and partially at sixother locations. These are marked in FIG. 16. Elimination of the threemajor 18 bp elements reduces expression to basal levels (Theilmann, D.A., and Stewart, S., Virology 187:84-96 (1992)). Primer extensionexperiments revealed that transcription initiates equally from eitherthe C or the A indicated. These two transcriptional start sites areadjacent to a CAGT sequence motif that has been shown to be conserved ina number of early genes (Blissard, G. W., and Rohrmann, G. F., Virology170:537-555 (1989)).

[0529] The GP64 promoter regulates expression of the baculovirus majorenvelope glycoprotein gene (GP64) of the budded virion. Studies haveshown that while the GP64 promoter is stimulated by the transcriptionaltransactivator IE-1, low levels of activity still occur withouttransactivation (Blissard, G. W., et al., Virology 190:783-793 (1992);Blissard, G. W., and Rohrrmann, G. F., J. Virology 65:5820-5827 (1991)).Furthermore, deletion analysis has identified the specific regionrequired for transcriptional initiation in the absence of IE-1(Blissard, G. W., et al., Virology 190:783-793 (1992); Blissard, G. W.,and Rohrmann, G. F., J. Virology 65:5820-5827 (1991)).

[0530] pIB/V5-His-DEST contains a 100 bp region of the Autographacalifornica nuclear polyhedrosis virus (AcMNPV) GP64 promoter which issufficient for activation of the blasticidin resistance gene (bsd) inthe absence of any baculovirus proteins. Using standard blasticidinconcentrations (10-80 μg/ml), stable transfectants will only be selectedif the bsd gene is expressed at suitable levels. Without wishing to bebound by theory, because of the minimal activity of the GP64 promoter,it is likely that only stable transfectants containing pIB/V5-His-DESTintegrated into the most transcriptionally active genomic loci will beselected. This allows generation of stable cell lines which will expresshigher levels of the protein of interest compared to cell linesexpressing the bsd gene product from the OpIE1 promoter, as in theparent pIB/V5-His vector.

[0531] Cell cultures of either Sf9 (catalog no. B82501, InvitrogenCorporation, Carlsbad, Calif.), Sf21 (catalog no. B82101, InvitrogenCorporation, Carlsbad, Calif.), or High Five™ cells (catalog no. B85502,Invitrogen Corporation, Carlsbad, Calif.) may be used in connection withthe present invention and may be grown and stored using conventionaltechniques well known in the art (e.g., Baculoviral Expression Systemsand Insect Cell Lines manual, Feb. 27, 2002, Invitrogen Corporation,Carlsbad, Calif.).

[0532] The pIB/V5-His-DEST vector is supplied as a supercoiled plasmid.Linearization of this vector is not required to obtain optimal resultsfor any downstream application. The vector may be resuspended at aconcentration of 50-150 ng/μl in sterile water, pH 8.0. To propagate andmaintain pIB/V5-His-DEST, Library Efficiency® DB3.1™ Competent Cells(Invitrogen Corporation, Carlsbad, Calif. Catalog no. 11782-018) may beused. The DB3.1™ E. Coli strain is resistant to CcdB effects and cansupport the propagation of plasmids containing the ccdB gene. Tomaintain integrity of the vector, select for transformants in mediacontaining 50-100 μg/ml ampicillin and 15 μg/ml chloramphenicol. The useof general E. coli cloning strains including TOP10 or DH5α is notrecommended for propagation and maintenance as these strains aresensitive to CcdB effects. In one alternative of this aspect of theinvention, the chloramphenicol resistance gene in the cassette can bereplaced by a spectinomycin resistance gene (see Hollingshead et al.,Plasmid 13(1):17-30 (1985), NCBI accession no. X02340 M10241), and thepcDNA destination vector containing attP sites flanking the ccdB andspectinomycin resistance genes can be selected onampicillin/spectinomycin-containing media. It has recently been foundthat the use of spectinomycin selection instead of chloramphenicolselection results in an increase in the number of colonies obtained onselection plates, indicating that use of the spectinomycin resistancegene may lead to an increased efficiency of cloning from that observedusing cassettes containing the chloramphenicol resistance gene.

[0533] To recombine a sequence of interest into pIB/V5-His-DEST, anentry clone containing the sequence of interest may be prepared. Acommercially available kit (e.g., the pENTR Directional TOPO® CloningKit, Invitrogen Corporation, Carlsbad, Calif. Catalog no. K2400-20,version B) can be used. Other suitable entry vectors are available fromInvitrogen Corporation, Carlsbad, Calif. Detailed information onconstructing an entry clone may be obtained from the manual providedwith the specific entry vector. For detailed information on performingthe LR recombination reaction, refer to the GATEWAY™ Technology manual,Invitrogen Corporation, Carlsbad, Calif.

[0534] A sequence of interest may contain a Kozak consensus sequencewith an ATG initiation codon for proper initiation of translation(Kozak, M., Nucleic Acids Res. 15:8125-8148 (1987); Kozak, M., J. CellBiology 115:887-903 (1991); Kozak, M., Proc. Natl. Acad. Sci. USA87:8301-8305 (1990)). An example of a Kozak consensus sequence isprovided below. Other sequences are possible, but the G or A at position−3 and the G at position +4 are the most critical for function (shown inbold). The ATG initiation codon is shown underlined.

[0535] (G/A)NNATGG

[0536] To include the V5 epitope and/or 6×His tag encoded by the vector,the sequence of interest may not contain a stop codon. A coding sequenceshould also be designed to be in frame with the C-terminal epitope tagafter recombination. To express a polypeptide with a native C-terminal(i.e., without the V5 epitope and/or 6×His tag), the sequence ofinterest should contain a stop codon in the entry clone.

[0537] Each entry clone contains attL sites flanking the sequence ofinterest. Sequences of interest in an entry clone may be transferred tothe destination vector backbone by mixing the DNAs with the GATEWAY™ LRClonase™ enzyme mix. The resulting LR recombination reaction may then betransformed into E. coli and the expression clone may be selected. In anembodiment, recombination between the attR sites on the destinationvector and the attL sites on the entry clone replaces the ccdB gene andthe chloramphenicol (Cm^(R)) gene with the sequence of interest andresults in the formation of attB sites in the expression clone.

[0538] The LR Clonase™ reaction; subsequent transformation of a suitableE. coli, and selection for an expression clone may be performed usingstandard techniques such as those provide in the GATEWAY™ Technologymanual.

[0539] The ccdB gene mutates at a very low frequency, resulting in avery low number of false positives. True expression clones will beampicillin-resistant and chloramphenicol-sensitive. Transformantscontaining a plasmid with a mutated ccdB gene will be both ampicillin-and chloramphenicol-resistant. A putative expression clone can be testedby growth on LB plates containing 30 μg/ml chloramphenicol. A trueexpression clone will not grow in the presence of chloramphenicol.

[0540] The recombination region of the expression clone resulting frompIB/V5-His-DEST×entry clone is shown in FIG. 17. Shaded regionscorrespond to those DNA sequences transferred from the entry clone intopIB/V5-His-DEST by recombination. Non-shaded regions are derived fromthe pIB/V5-His-DEST vector. The underlined nucleotides flanking theshaded region correspond to bases 609 and 2292, respectively, of thepIB/V5-His-DEST vector sequence.

[0541] To confirm that a coding sequence on the sequence of interest isin frame with the C-terminal V5 epitope and polyhistidine tag, theexpression construct may be sequenced, for example, using the OpIE2Forward and Reverse primer sequences. Refer to FIG. 17 for the sequenceand location of the primer binding sites.

[0542] Plasmid DNA for transfection into insect cells must be very cleanand free from phenol and sodium chloride. Contaminants will kill thecells, and salt will interfere with lipid complexing, decreasingtransfection efficiency. The expression construct plasmid may beprepared using standard techniques, for example, columnchromatography(e.g., the S.N.A.P.™ MiniPrep Kit Catalog no. K1900-01,Invitrogen Corporation, Carlsbad, Calif.). Typical yields of plasmidusing this technique are 10-15 μg of plasmid DNA from 10-15 ml ofbacterial culture. Plasmid can be used directly for transfection ofinsect cells.

[0543] One technique suitable to introduce the nucleic acid molecules ofthe invention into host cells is lipid-mediated transfection (e.g.,using Cellfectin® Reagent, catalog no. 10362010, Invitrogen Corporation,Carlsbad, Calif.). Other lipids may be substituted, althoughtransfection conditions may have to be optimized. Expected TransfectionEfficiency using Cellfectin® Reagent: 40-60% for Sf9 or Sf21 cells and40-60% for High Five™ cells. Other transfection methods (e.g., calciumphosphate and electroporation (Mann and King, 1989)) may also be usedwith High Five™ cells.

[0544] Controls may be included in the transfection reaction, forexample, IB/V5-His-GW/lacZ vector as a positive control for transfectionand expression and lipid only as a negative control DNA only to checkfor DNA contamination.

[0545] pIB/V5-His-GW/lacZ is provided as a positive control vector fortransfection and expression (see FIG. 18 for a map). The vector allowsexpression of a C-terminally tagged β-galactosidase fusion polypeptidethat may be detected by Western blot or functional assay.pIB/V5-His-GW/lacZ is a 6478 bp control vector containing the gene forβ-galactosidase. pIB/V5-His-GW/lacZ was constructed using the GATEWAY™LR recombination reaction between an entry clone containing the lacZgene and pIB/V5-His-DEST. β-galactosidase is expressed as a fusion tothe C-terminal tag. The molecular weight of the fusion polypeptide isapproximately 120 kDa.

[0546] To propagate and maintain the plasmid: resuspend the vector in 10μl sterile water to prepare a 1 μg/μl stock solution and use the stocksolution to transform a recA, endA E. coli strain like TOP10, DH5a,JM109, or equivalent. Select transformants on LB agar plates containing50-100 μg/ml ampicillin. Optionally, a glycerol stock of a transformantcontaining plasmid may be prepared for long-term storage.

[0547] For each transfection, log-phase cells with greater than 95%viability may be used. A time course for expression of the sequence ofinterest may be performed. For example, expression of a polypeptideencoded by the sequence of interest may be assayed for at 2, 3, and 4days post transfection. One or more 60 mm plate may be used for eachtime point. For Sf9, Sf21, or High Five™ cells, 1×10⁶ cells may beseeded in appropriate serum-free medium in a 60 mm dish. Rock gentlyfrom side to side for 2 to 3 minutes to evenly distribute the cells.Cells may be 50 to 60% confluent.

[0548] Incubate the cells for at least 15 minutes without rocking toallow the cells to fully attach to the bottom of the dish to form amonolayer of cells.

[0549] Verify that the cells have attached by inspecting them under aninverted microscope.

[0550] Nucleic acid molecules of the invention may be introduced intohost cells using standard techniques. A protocol for use of Cellfectin®Reagent is provided below. Other conditions for transfection may beempirically determined by one skilled in the art using routineexperimentation. Preferably, a plasmid is not linearized prior tointroduction into a host cell. Linearizing a plasmid appears to decreaseprotein expression. The reason for this is not known.

[0551] A suitable transfection may employ: 1-10 μg of purifiedpIB/V5-His-DEST expression construct (˜1 μg/μl in TE buffer); eitherlog-phase Sf9 or Sf21 cells (1.6-2.5×10⁶ cells/ml, >95% viability) orlog-phase High Five™ cells (1.8-2.3×10⁶ cells/ml, >95% viability),growing in serum-free medium (e.g., Grace's Medium without supplements;serum-free medium 60 mm tissue-culture dishes; 1.5 ml sterilemicrocentrifuge tubes; rocking platform only (NOT orbital); 27° C.incubator; inverted microscope; paper towels and air-tight bags orcontainers; and 5 mM EDTA, pH 8.

[0552] Transfection may comprise mixing plasmid DNA and Cellfectin® inan appropriate medium and incubating with freshly seeded insect cells.The amount of cells, liposomes, and plasmid DNA described herein hasbeen optimized for 60 mm culture plates. Other transfection conditionsmay be used with other size plates or flasks. Optimizing conditions forother volumes of transfection may be accomplished by one skilled in theart using routine experimentation. Serum-free medium (e.g., Sf-900 IISFM (catalog no. 1090207) to transfect Sf9 or Sf21 cells and ExpressFive® SFM (catalog no. 10486017) to transfect High Five™ cells,available from Invitrogen Corporation, Carlsbad, Calif.) can be used.Grace's Medium without supplements may also be used. The proteins in theFBS and supplements will interfere with the liposomes, causing thetransfection efficiency to decrease.

[0553] To prepare each transfection mixture, a 1.5 ml microcentrifugetube may be used. The following reagents may be added: 1 ml of Grace'sMedium OR appropriate serum-free medium; 1-10 μl nucleic acid moleculeof the invention (e.g., pIB/V5-His plasmid or construct) at aconcentration of ˜1 μg/μl in TE, pH 8; 20 μl Cellfectin® Reagent (mixedwell before use and always added last). The transfection mixture may bemixed gently for 10 seconds and incubated at room temperature for 15minutes.

[0554] The medium covering the cells to be transfected should be removedwithout disrupting the monolayer. If the medium contained serum, washthe cells by carefully adding 2 ml of fresh Grace's Medium withoutsupplements or FBS to remove trace amounts of serum that will decreasethe efficiency of liposome transfection and remove the wash.

[0555] The entire transfection mix described above may be added dropwiseinto the 60 mm dish. The drops may be evenly distributed over themonolayer. This method reduces the chances of disturbing the monolayer.Repeat for all transfections.

[0556] The dishes may be incubated at room temperature for 4 hours on aside-to-side, rocking platform. A suitable speed for the platform is ˜2side to side motions per minute. Instead of a platform rocker, thedishes may be manually rocked periodically.

[0557] Following the 4-hour incubation period, 1-2 ml of complete TNM-FHmedium (Sf9 or Sf21 cells) or the appropriate serum-free medium (Sf9,Sf21, or High Five™ cells) may be added to each 60 mm dish. The dishesmay be placed in a sealed plastic bag with moist paper towels to preventevaporation and incubated at 27° C. It is not necessary to remove thetransfection solution as Cellfectin® Reagent is not toxic to the cells.If a different lipid is used and loss of viability is observed, thenremove the transfection solution after 4 hours, rinse twice with medium,and replace with 1-2 ml of fresh medium.

[0558] The cells may be harvested, for example, at 2, 3, and 4 days posttransfection and assayed for expression of the sequence of interest.Additional fresh medium need not be added to the cells if the cells aresealed in an airtight plastic bag with moist paper towels.

[0559] Expression of a sequence of interest from the expression clonecan be performed in transiently transfected cells or stable cell lines.A sample protocol to detect by Western blot a polypeptide encoded by asequence of interest expressed as a fusion polypeptide is providedbelow.

[0560] The cells from one 60 mm plate may be used for each expressionexperiment. A suitable cell lysis buffer may be used. One suitablebuffer is 50 mM Tris, pH 7.8, 150 mM NaCl, 1% Nonidet P-40.

[0561] The medium may be removed from the cells. If the polypeptideexpressed from the sequence of interest is predicted to be secreted,save and assay both the medium and the cell pellet. Cell lysis buffer,100 μl, may be added to the plate and the cells may be sloughed orscraped into a microcentrifuge tube. The cells may be vortexed to ensurethey are completely lysed. The lysed cells may be centrifuged at maximumspeed in a microfuge for 1-2 minutes to pellet nuclei and cellmembranes. The supernatant may be transferred to a new tube. If amembrane protein is expressed from the sequence of interest, it may belocated in the pellet. The pellet and the lysate may be assayed. Theprotein concentration in the lysate may be determined, for example, bythe Bradford, Lowry, or BCA assays (Pierce).

[0562] Samples may be mixed with SDS-PAGE sample buffer as follows: 30μl lysate with 10 μl 4×SDS-PAGE sample buffer; the pellet may beresuspended in 100 μl 1×SDS-PAGE sample buffer; 30 μl medium may bemixed with 10 μl 4×SDS-PAGE sample buffer. Because of the volume ofmedium, it is difficult to normalize the amount loaded on an SDS-PAGEgel. Optionally, the medium may be concentrated to facilitatenormalization. Samples may be boiled for 5 minutes, centrifuged briefly,and approximately 3 to 30 μg protein loaded per lane of an SDS-PAGE gel.The same volume of sample may be added for both the pellet sample andthe lysate sample. The amount to load may be determined by one skilledin the art using routine experimentation. Samples may be separated byelectrophoresis, blotted, and probed with a suitable antibody usingstandard techniques.

[0563] A polypeptide expressed from a sequence of interest as a fusionpolypeptide may be detected by Western blot analysis, for example, withthe Anti-V5 antibodies or the Anti-His(C-term) antibodies available fromInvitrogen Corporation, Carlsbad, Calif. or an antibody thatspecifically recognizes the polypeptide. In addition, the Positope™Control Protein (Invitrogen Corporation, Carlsbad, Calif., Catalog no.R900-50) is available for use as a positive control for detection offusion proteins containing a V5 epitope or a 6×His tag.

[0564] If the pIB/V5-His-GW/lacZ plasmid is used as a positive controlvector, β-galactosidase expression may be assayed by Western blotanalysis or activity assay (Miller, J. H., Experiments in MolecularGenetics, Cold Spring Harbor Laboratory, Cold Spring Harbor, N.Y.(1972)). Commercially available antibodies (e.g., InvitrogenCorporation, Carlsbad, Calif., β-Gal Antiserum, Catalog no. R901-25), orassay kits (e.g., Invitrogen Corporation, Carlsbad, Calif. β-Gal AssayKit, Catalog no. K1455-01 and β-Gal Staining Kit Catalog no. K1465-01)may be used for detection of β-galactosidase expression.

[0565] The C-terminal peptide containing the V5 epitope and thepolyhistidine tag will add approximately 5 kDa in molecular weight to apolypeptide expressed from a sequence of interest.

[0566] Selecting Stable Cell Lines

[0567] Stable expression cell lines can be created for long-term storageand large-scale production of the desired polypeptide. Note that stablecell lines are created by multiple copy integration of the vector.Amplification as in the case with calcium phosphate transfection andhygromycin resistance in Drosophila is generally not observed.

[0568] Blasticidin may be used to select for stably transformed cells.Gloves, mask, goggles, and protective clothing (e.g. a laboratory coat)should be worn when handling blasticidin. Weighing blasticidin andpreparing solutions should be done in a hood. Blasticidin may beinactivated for disposal by adding sodium bicarbonate. Blasticidin issoluble in water and acetic acid. Water is generally used to preparestock solutions of 5 to 10 mg/ml. Blasticidin may be dissolved insterile water and filter-sterilized. Blasticidin is unstable insolutions with a pH greater than 8.0. The pH of a solution ofblasticidin may be 7.0. Blasticidin solutions may be divided intoaliquots in small volumes and frozen at −20° C. for long-term storage orstored at +4° C. for short term storage. Aqueous stock solutions arestable for 1-2 weeks at +4° C. and 6-8 weeks at −20° C. Stock solutionsshould not be subjected to multiple freeze/thaw cycles (do not store ina frost-free freezer). Solutions should be discarded after 1-2 weeksstorage at +4° C.

[0569] Cytopathic effects should be visible within 3-5 days depending onthe concentration of blasticidin in the medium. Sensitive cells willenlarge and become filled with vesicles. The outer membrane will showsigns of blebbing, and cells will eventually detach from the plate.Blasticidin-resistant cells should continue to divide at regularintervals to form distinct colonies. There should not be any distinctmorphological changes between blasticidin-resistant cells compared tocells not under selection with blasticidin.

[0570] In general, concentrations of blasticidin around 10 μg/ml willkill Sf9 or Sf21 cells (in complete TNM-FH medium) and concentrationsaround 20 μg/ml will kill High Five™ cells (in Express Five® SFM) withinone week, although a few cells may remain that exclude trypan blue. Toobtain faster and more thorough killing, 50-80 μg/ml blasticidin may beused. Once blasticidin-resistant clones have been obtained, cells may bemaintained in lower concentrations of blasticidin (e.g., 10-20 μg/ml).An appropriate concentration of blasticidin for any specific cell typemay be determined by one skilled in the art by performing a kill curve.

[0571] A suitable protocol for establishing a kill curve is provided.Assays may be conducted in 24-well tissue culture plates. Suitablemedium (e.g., TNM-FH medium or the serum-free medium of choice) may beprepared and supplemented with concentrations ranging from 0 to 100μg/ml blasticidin. Generally, concentrations that effectively killlepidopteran insect cells within a week are in the 50 to 80 μg/ml range.While 10-20 μg/ml blasticidin will kill cells within a week, higherconcentrations will result in faster and more thorough killing. Inaddition, using higher concentrations of blasticidin may result inenrichment of clones containing multiple integrations of a sequence ofinterest. Test varying concentrations of blasticidin on a cell line ofinterest to determine the concentration that kills the cells within aweek (kill curve). The concentration of drug that kills the cells ofinterest within a week should be used.

[0572] To isolate a stable cell line, a mock transfection and a positivecontrol (e.g., pIB/V5-His-GW/lacZ) may be used. Cells may be transfectedas described above. Forty-eight hours post transfection, thetransfection solution may be removed and fresh medium containing noblasticidin may be added. The cells may be split 1:5 (20% confluent) andallowed to attach overnight before adding selective medium. The mediummay be removed and replaced with medium containing blasticidin at theappropriate concentration. The cells may be incubated at 27° C. Theselective medium may be replaced every 3 to 4 days until foci areobserved. Cloning cylinders or limiting dilution may be used to isolateclonal cell lines. Optionally, resistant cells may be allowed tocontinue grow out to confluence for a polyclonal cell line (2 to 3weeks).

[0573] A polyclonal cell line may be isolated by allowing the resistantcells grow to confluence and splitting the cells 1:5. The polyclonalcell line may be tested for expression. Medium without blasticidinshould be used when splitting cells and cells should be allowed toattach before adding selective medium.

[0574] Resistant cells may be expanded into flasks to prepare frozenstocks. Medium containing blasticidin should be used when maintainingstable lepidopteran cell lines. The concentration of blasticidin may belowered to 10 pg/ml for maintenance.

[0575] Isolation of Clonal Cell Lines Using Cloning Cylinders

[0576] Multiple foci may be isolated for expression testing. As inmammalian cell culture, the location of integration may affectexpression of a sequence of interest. Selections may be performed insmall plates or wells. Cells should not be allowed to dry out during theselection.

[0577] The closed plate may be examined under a microscope and thelocation of one or more colony marked on the top of the plate. Themarkings may then be transferred to the bottom of the plate. Orientationmarks may be included. Each colony may contain 50 to 200 cells. Sf9cells tend to spread more than High Five™ cells. The culture dish may bemoved to a sterile cabinet and the lid removed. A thin layer of sterilesilicon grease may be applied to the bottom of a cloning cylinder(Scienceware, Catalog no. 378747-00 or Belco, Catalog no. 2090-00608),using a sterile cotton-tipped wooden applicator. The layer should bethick enough to retard the flow of liquid from the cylinder, withoutobscuring the opening on the inside. Cloning cylinders and silicongrease can be sterilized together by placing a small amount of grease ina glass petri dish and placing the cloning cylinders upright in thegrease. After autoclaving, the grease will have spread out in a thinlayer to coat the bottom of the cylinders.

[0578] The culture medium may be removed and the cylinder placed firmlyand directly over the marked area. A microscope may be used to directplacement of the cylinder. 20 to 100 μl of medium (no blasticidin) maybe used to dislodge the cells. The cells and medium may be removed andtransferred to a microtiter plate and the cells may be allowed toattach. The medium may be removed and replaced with selective medium forculturing. The cell line may be expanded and tested for expression ofthe sequence of interest.

[0579] Isolation of Clonal Cell Lines Using a Dilution Method

[0580] Clonal cell lines may be established using a dilution method. Theobjective of this method is to dilute the cells so that under selectivepressure only one stable viable cell per well is achieved. The highertransfection efficiency, the more the cells should be diluted. Theprotocol below works well with cells transfected at 5-10% efficiency.

[0581] Forty-eight hours after transfection, cells may be diluted to1×10⁴ cells/ml in medium without blasticidin. Other dilutions of theculture may also be used as transfection efficiency will determine howmany transformed cells there will be per well. 100 μl of the cellsolution may be added to 32 wells of a 96-well microtiter plate (8 rowsby 4 columns). The remaining cells may be diluted 1:1 with mediumwithout blasticidin and add 100 μl of this solution added to the nextgroup of 32 wells (8×4). The remaining cells may be diluted 1:1 withmedium without blasticidin and 100 μl of this solution added to the lastgroup of 32 wells. Although the cells can be diluted to low numbers,cell density is critical for viability. If the density drops below acertain level, the cells will not grow.

[0582] The cells may be allowed to attach overnight, then the mediumremoved and replaced with medium containing blasticidin. Removing andreplacing medium may be tedious. Optionally, it is possible to dilutethe cells directly into selective medium if they are handled gently.

[0583] The plate may be wrapped and incubated at 27° C. for 1 week. Itis not necessary to change the medium or place in a humid environment.The plate may be checked after a week and the wells that have only onecolony may be marked. The plate may be incubated until the colony fillsmost of the well. The cells may be harvested and transferred to a24-well plate with 0.5 ml of fresh medium containing blasticidin. Theclone may be expanded to 12- and 6-well plates, and finally to a T-25flask.

[0584] Each cell line may be assayed for yield of the desiredpolypeptide and the one with the highest yield may be scaled-up and usedfor purification of recombinant polypeptide. For secreted polypeptides,the cell pellet as well as the medium may be assayed. The yield ofpolypeptide in the cells may be compared to the yield of polypeptide inthe medium.

[0585] Master stocks and working stocks of stable cell lines may beprepared prior to scale-up and purification.

[0586] Purification

[0587] A polypeptide expressed from a sequence of interest may bepurified using standard techniques. Stable cell lines prepared asdescribed above may be expanded into larger flasks, spinners, shakeflasks, or bioreactors to obtain the desired yield of polypeptide. If apolypeptide expressed from a sequence of interest is secreted, cells maybe cultured in serum-free medium to simplify purification.

[0588] A 6His tagged fusion polypeptide may be purified using theProBond™ Purification System, the Ni-NTA Purification System, or asimilar product. Both purification systems contain a metal-chelatingresin specifically designed to purify 6×His-tagged polypeptides.

[0589] Cells may be maintained in a medium having a concentration ofblasticidin of 10 μg/ml. Cells may be switched from complete TNM-FHmedium to serum-free medium during passage.

[0590] Adding serum-free medium directly to a metal-chelating resin suchas ProBond™ to purify a secreted polypeptide from serum-free medium willstrip the nickel ions from the resin. To purify 6×His-tagged recombinantpolypeptides from the culture medium, dialysis or ion exchangechromatography may be performed prior to affinity chromatography onmetal-chelating resins. Dialysis allows removal of media components thatstrip Ni⁺² from metal-chelating resins. Ion exchange chromatographyallows removal of media components that strip Ni⁺² from metal-chelatingresins and concentration of sample for easier manipulation in subsequentpurification steps.

[0591] Conditions for successful ion exchange chromatography will varydepending on the polypeptide. For more information, refer to Coligan, J.E., et al., Current Protocols in Protein Science, Chanda, V. B., ed.,John Wiley and Sons, Inc., New York (1998), Ausubel, F. M., et al.,Current Protocols in Molecular Biology, Unit 10 (1994), or Deutscher, M.P., “Guide to Protein Purification,” in Methods in Enzymology, Vol. 182,Simon, M. I., ed., Academic Press, San Diego, Calif. (1990).

[0592] Many insect cell proteins are naturally rich in histidines, withsome containing stretches of six histidines. When using the ProBond™Purification System or other similar products to purify 6×His-taggedpolypeptides, these histidine-rich polypeptides may co-purify with apolypeptide of interest. The contamination can be significant if thepolypeptide of interest is expressed at low levels. 5 mM imidazole maybe added to the binding buffer prior to addition of the polypeptidemixture to the column. Addition of imidazole may help to reducebackground contamination by preventing polypeptides with low specificityfrom binding to the metal-chelating resin.

[0593] If the polypeptide of interest is 6×His-tagged and expressedintracellularly, the cells may be lysed and the lysate added directly tothe ProBond™ column. 5×10⁶ to 1×10⁷ cells may be used for purificationof a polypeptide of interest on a 2 ml ProBond™ column (see ProBond™Purification System manual, catalog nos. R801-01, R801-15, version F,Invitrogen Corporation, Carlsbad, Calif.).

[0594] A suitable protocol is to seed 2×10⁶ cells in two or three 25 cm²flasks, grow the cells in selective medium until they reach confluence(4×10⁶ cells); wash cells once with PBS (Phosphate Buffered Saline, pH7.4; Invitrogen Corporation, Carlsbad, Calif. Catalog no. 10010-023);harvest the cells by sloughing; transfer the cells to a sterilecentrifuge tube; and centrifuge the cells at 1000×g for 5 minutes. Thecells may be lysed immediately or frozen in liquid nitrogen and store at−80° C. until needed.

[0595] Many protocols are suitable for purifying polypeptides from themedium. The choice of protocol depends on the nature of the polypeptidebeing purified. The culture volume needed to purify sufficientquantities of polypeptide is dependent on the expression level of thepolypeptide and the method of detection. One skilled in the art candevelop suitable purification protocols using routine experimentation.

Example 6 Construction of Recombinant Baculoviruses

[0596] Baculoviruses have been extremely useful tools for heterologousexpression of proteins in insect cells. Improved methods for cloninggenes into baculoviral genomes (e.g., the 134 kb AcMNPV genome) havegreatly simplified the process of recombinant baculovirus construction;however obtaining a purified viral stock still requires plaquepurification and a minimum of 10-14 days. Current methods rely onrecombination in insect or bacterial cells and are not well adapted forhigh-throughput experiments. To meet these challenges, materials andmethods of the invention permit the construction of recombinantbaculovirus in vitro. The recombinant baculovirus may be transfecteddirectly into insect cells to generate the baculovirus stock.

[0597] A baculovirus genome containing a recombination cassette (DEST)bounded by attR recombination sites compatible with GATEWAY™ entryvectors (Invitrogen Corporation, Carlsbad, Calif.) was constructed. Twotransposition cassettes were constructed one with and one without themellitin leader sequence. A schematic representation of the cassettewithout the mellitin sequence is provided in FIG. 19A and the sequenceis provided in Table 13. A schematic representation of the cassette withthe mellitin sequence is provided in FIG. 19B and the sequence isprovided in Table 14. The DEST cassettes contain the HSV thymidinekinase (TK) gene driven by an immediate early promoter (IE-0 promoter)and the lacZ gene driven by a late promoter (P10 promoter). The genespermit identification of non-recombinant virus using a blue whitescreening protocol and selection against non-recombinant viruses usingganciclovir. The cassettes also contain the V5 epitope and a 6-Histidinesequence outside the attR2 recombination site. The sequence of thecassette contains a recognition site for the restriction enzyme Bsu36I(and its isoschizomer AocI) that is used to linearize the viral genome.

[0598] The cassette may be inserted into a baculoviral genome such thata sequence of interest in the Entry Clone may be operably linked to abaculoviral promoter (e.g., the polyhedrin promoter (ph pr in FIG. 20))upon insertion of the sequence of interest into the viral genome. Inpractice, any eukaryotic cellular or viral promoter can be used toexpress a gene introduced from an entry clone, e.g. promoters from anyof the above named baculovirus species, whether they are early, late, orvery late. Although depicted as a gene sequence in FIG. 20, any sequenceof interest may be inserted; the present invention is not limited tosequences encoding polypeptides.

[0599] In one embodiment, the nucleic acid sequence of interest may berecombined directly into the baculovirus genome downstream of thepolyhedrin promoter, replacing the TK and lacZ genes. With reference toFIG. 20, the linearized baculoviral genome is depicted as a gappedcircle. In the presence of the appropriate recombination proteins, therecombination sites (e.g., attR1 and attR2 sites) on the baculoviralgenome will recombine with the recombination sites (e.g., attL1 andattL2 sites) on the nucleic acid molecule comprising the sequence ofinterest (Entry Clone in FIG. 20) resulting in recircularization of thebaculoviral genome. The recombination reaction results in the transferof the sequence of interest (depicted as a gene of interest (GOI) inFIG. 20) into the baculoviral genome. The transfer also results in theexcision of the portions of the baculoviral genome between the attRrecombination sites.

[0600] The resultant DNA may be directly transfected into insect cellsto produce the recombinant viral stock. When the cells are grown onganciclovir, only recombinant virus is able to replicate; replication ofparental virus is prevented because of the TK gene product. Thedestination cassette may also be placed under the control of the CMVpromoter or other promoter active in mammalian cells, for the purpose oftransducing mammalian cells using baculovirus.

[0601] To demonstrate the feasibility of this system and to optimizeconditions, the GFP coding sequence was first cloned into a nucleic acidmolecule between two recombination sites and then transferred usingrecombinational cloning into a baculovirus genome comprising twocompatible recombination sites. Sf21 cells were transfected with therecombination reaction mixture. After three days, the media from thesecells containing budded virus produced from the first rounds ofreplication was used to infect a second population of cells, this timegrown under ganciclovir selection. After 4 days, these cells wereexamined for GFP fluorescence and stained for LacZ expression. Cellsinfected by recombinant virus expressing GFP were fluorescent, whilecells infected with remaining parental virus stained positive for LacZexpression. Using this assay method, conditions for transfection andganciclovir counter selection were optimized. Under ideal conditions,small scale virus stocks essentially free of parental virus wereproduced within 7 days post-transfection. These stocks are suitable forcreation of high titer stocks or further expression studies.

[0602] The utility of this system was then demonstrated for use in a 96well format with collections of genes cloned into an Entry vector.Multiple genes in 96 well plates were cloned and screened for expressionin parallel. Within seven days, purified viral stocks were available forscale-up or further expression studies.

[0603] In some embodiments, the present invention provides a new methodfor baculovirus cloning based on lambda recombination that is faster,requires less hands-on time, is more reliable, and is suitable for highthroughput expression in 96 well plates.

[0604] In some embodiments, the present invention provides isolatednucleic acids comprising nucleic acid sequences that function aspromoters. Optionally, the nucleic acid molecules may comprise one ormore sequences of interest (e.g., ORFs, etc.) operably linked to one ormore of the nucleic acid sequences that function as promoters. Thesepromoters may function in any cell type, for example, mammalian, insect,etc.

[0605] In some embodiments, the promoters are tightly regulated. Forexample, in some embodiments, the promoters are not active unless one ormore transactivators are present. In some embodiments, the nucleic acidsequences that function as promoters include, but are not limited to,the AcMNPV ORF 25 promoter sequence, the AcMNPV lef 3 promoter sequence,the AcMNPV TLP promoter sequence, the AcMNPV homologous repeat 5sequence, other baculovirus homologous repeat sequences, and the like.The nucleic acid sequences of the AcMNPV ORF 25 promoter sequence, theAcMNPV lef 3 promoter sequence, the AcMNPV TLP promoter sequence, andthe AcMNPV homologous repeat 5 sequence are provided in Table 15.

[0606] In some embodiments, the promoters discussed above are not activeunless one or more transactivators are present. One suitabletransactivator is the baculoviral IE-1 protein. The IE-1 promotersequence, coding sequence, and polypeptide sequence are provided inTable 16. The transactivator may be provided on the same nucleic acidmolecule comprising the promoter sequence or on another nucleic acidmolecule (e.g., plasmid, virus, host cell genome, etc.). In someembodiments, the promoter sequence operably linked to a sequence ofinterest may be on one nucleic acid molecule (e.g. a plasmid) and thetransactivator sequence may be on a different nucleic acid molecule(e.g., a virus such as a baculovirus). The nucleic acid moleculecomprising the promoter sequence operably linked to a sequence ofinterest may be introduced into a host cell, for example, bytransfection. The sequence of interest is not expressed or issubstantially not expressed in the absence of a transactivator. In someembodiments, the host cell may be a eukaryotic cell, for example, amammalian cell or an insect cell. The host cell comprising the nucleicacid molecule comprising the promoter sequence operably linked to asequence of interest may be further contacted with a second nucleic acidmolecule comprising the a sequence encoding the transactivator. Uponexpression of the transactivator, the sequence of interest is expressed.In some embodiments, the transactivator polypeptide may be directlytransfected into cells comprising the nucleic acid molecule comprisingthe promoter sequence operably linked to a sequence of interest. Suchtransactivator polypeptides may be present as native polypeptides or asfusion polypeptides, for example, as fusions with the herpesvirus VP22polypeptide.

[0607] Nucleic acid molecules comprising the promoters discussed abovemay be used to conditionally express any sequence of interest. In someembodiments, the sequence of interest may encode a toxic polypeptide.

[0608] In some embodiments, nucleic acid molecules comprising thepromoter sequences described above may have a homologous repeat (hr)sequence in cis with the promoter. Such homologous repeat sequences maybe required for hr-dependent IE-1 transactivation.

[0609] The sequences provided in Table 15 are capable of functioning asconditionally activated promoters. The present invention also comprisesportions of the sequences of Table 15 that function as conditionallyactive promoters. Such promoters may be activated by the IE-1polypeptide. Such portions may comprise at least 50%, 60%, 70%, 80%,90%, 95%, or more of one or more of the sequences in Table 15.

Example 7

[0610] In some embodiments, materials and methods of the invention maybe used to create stable cell lines expressing a nucleic acid sequenceof interest. One non-limiting example is the InsectSelect™ system(Invitrogen Corporation, Carlsbad, Calif.), which is a stable insectcell expression system that utilizes a single plasmid for expression andselection. Nucleic acid molecules of the invention (e.g., InsectSelect™vectors) may utilize different baculovirus immediate early promoters forexpression of a sequence of interest and a selectable marker. Nucleicacid molecules of the invention may be constructed to be used inrecombinational cloning methods. For example, pIB/V5-His (catalog no.V802001, Invitrogen Corporation, Carlsbad, Calif.) has been modified forusing in methods involving recombinational cloning (e.g., GATEWAY™cloning). In the modified vector, a different promoter is used to drivetranscription of the blasticidin resistance gene than the OpIE-1promoter used in pIB/V5-HIS.

[0611] The OpIE-1 promoter was replaced with long or short versions ofAcMNPVgp64 or pe38 promoters, using a Topoisomerase I mediated ligationstrategy (FIG. 21). The AcMNPV gp64 and pe38 promoters were amplifiedfrom cosmid #58 (comprising AcMNPV bases 99803-132856 from a cosmidlibrary of the AcMNPV genome, Harwood et al. Virology. 250:113-134,1998) with promoter-specific primers that were appended at their 5′ endswith antisense TOPO sites and six additional bases (FIG. 21). pIB/V5-Hiswas amplified with primers that included an anti-sense topoisomerasesite and a six base sequence that becomes an overhang followingtopoisomerase binding. Each promoter (gp64s is illustrated) wasamplified with similarly designed primers. Following binding, theoverhangs annealed and were ligated by the enzyme. The oligonucleotidesequences are given below. The antisense topoisomerase sites areunderlined. 17852 pIB Neg For TGAGTCAAGGGCTGCCGGGCTGCAGCACTG 17853 pIBNeg Rev CGGAACAAGGGCATGACCAAAATCCCTTAACG 17849 gp64 ForGACTCAAAGGGCTTGCTTGTGTGTTCCTTATTG 17850 gp64s RevGTTCCGAAGGGTTGTGTCACGTAGGCCAGATAAC 17851 gp64L RevGTTCCGAAGGGAATAATCGATTTAAGGGTGTAATACTC 17857 pe38 ForGACTCAAAGGGTTTGCTTATTGGCAGGCTCTCC 17858 pe38s RevGTTCCGAAGGGTATCTGTCCCCCACTCAGGC 17859 pe38L RevGTTCCGAAGGGTAAAGTTGATGCGGCGACGGC

[0612] The pIB/V5 His backbone was amplified using similarly designedprimers. The PCR products were purified by gel electrophoresis and SNAPmini-prep columns. Following DpnI treatment to eliminate residualtemplate vector, the PCR products were repurified by SNAP minipreps,eluted in 30 μl water and joined using topoisomerase (FIG. 21).Topoisomerase reactions were incubated at room temperature for 10 minand contained 8 μl of each PCR product, 50 mM Tris, pH 7.5, 0.1 μg/μlenzyme in 20 μl total volume. TOP10 E. Coli were transformed with thejoined PCR products. Following selection on ampicillin plates, resultingcolonies were grown overnight, and plasmid DNA isolated by miniprep(SNAP). The presence of the promoters was confirmed by restrictiondigest analysis. The construct containing gp64s was ultimately chosenfor GATEWAY™ adaptation (see below).

[0613] pIB/V5-His gp64 was modified to comprise recombination sites(i.e., GATEWAY™ adapted) by cloning a HindIII/XbaI fragment from pDEST38into pIB/V5-His gp64, cut with the same enzymes. The vector was fullysequenced. A plasmid map is provided (FIG. 22).

[0614] To test the modified vector in a recombinational cloningreaction, pIB/V5-His gp64Dest was used for LR reactions with attL entryvectors containing LacZ, Calmodulin, TFIIS, and Apolipoprotein. Theprotocol used differed slightly from the protocol suggested in theGATEWAY™ manuals. The reaction conditions used were as follows:

[0615] 2 μL LR clonase enzyme mix (catalog no. 11791043, InvitrogenCorporation, Carlsbad, Calif.)

[0616] 2 μL LR reaction buffer

[0617] 1 μL pENTR clone (˜300 ng DNA)

[0618] 1 μL pDEST vector (˜300 ng DNA)

[0619] 4 μL 0.5 M Tris buffer (pH 7.5)

[0620] Recombination reactions were incubated for 3 h at roomtemperature.

[0621] Reactions were not proteinase K treated. 2 μl of eachrecombination reaction was used to transform 50 μl TOP10 chemicallycompetent bacteria. Half of the transformation mix was plated andyielded an average of 230 colonies. Thus, approximately 8000 colonieswere obtained per μg entry vector. Colonies were grown in LB/Ampovernight and DNA was isolated by SNAP miniprep.

[0622] Experiments were performed with Sf21 cells or HighFive cells inserum-containing or serum free media (SFM). Grace's supplemented mediawith 10% FBS was used for both Sf21 and HighFive cells. For SFMtreatments, Sf900II or ExpressFive media were used for Sf21 or HighFivecells, respectively. Twenty-four well plates were seeded with 1.8×10⁵cells per well, and after 1 h attachment, washed with Grace'sunsupplemented media. Transfection mixes contained 0.2 μg DNA and 1 μlCellfectin® in 40 μl Grace's unsupplemented media and incubated for 30min at RT. The transfection mixture was then diluted to 200 μl finalvolume in Grace's unsupplemented media and added to each well. Cells andtransfection mix were incubated for 5 h with gentle rocking after whichthe mix was replaced with the appropriate media as described above. 48 hlater the media was replaced with the same media containing between 10and 25 μg/μl blasticidin, depending on the experiment. Cells used fromstable cultures were under selection for at least 7 days. Cells weresplit as needed to maintain log-phase growth. Typically, 10 μg/mlblasticidin may be used for general purposes. However, one skilled inthe art can optimize selection parameters for each construct using onlyroutine experimentation.

[0623] Protein expression was monitored by western blot or LacZ activityassays. Cells from six well plates (approximately 10⁶ per well) werewashed 2× in PBS, transferred to 1.7 ml tubes, spun down, resuspended in500 μl lysis buffer (Tropix Galacto light kit, catalog no. T1006,Applied Biosystems, Foster City, Calif.), and then subjected to twofreeze-thaw cycles. Lysates were microfuged at 16,000×g for 5 min.Supernatants were stored at −20° C. until used. Lysate proteinconcentration was measured using the BioRad protein assay against BSA asa standard. Various amounts of protein were denatured in LDS samplebuffer (catalog no. NP0008, Invitrogen Corporation, Carlsbad, Calif.)and loaded on 4-12% NuPAGE gels (Invitrogen Corporation, Carlsbad,Calif.). Following electrophoresis, proteins were transferred to PVDF.The Western Breeze kit (catalog no. WB7104, Invitrogen Corporation,Carlsbad, Calif.) was used to visualize protein bands using anti-V5coupled alkaline phosphatase at a 1:5000 dilution unless notedotherwise.

[0624] Without being bound by theory, is was though that use of a weakerpromoter to drive antibiotic resistance would result in stable culturesthat expressed the gene of interest at higher levels because the bsdgene (blasticidin resistance gene) was expressed at a lower level,integration of the plasmid containing the bsd gene would occur in moreloci or in loci that were transcriptionally more active. Transcriptionof many baculovirus genes has been characterized, and suitable promoterswere selected. The gp64 and pe38 promoters have both been extensivelystudied (Friesen, Regulation of baculovirus early gene expression, p.141-170. In The Baculoviruses. L. K. Miller (ed.), Plenum Press, NewYork., 1997). The pe38 promoter is an immediate early promoter and thusdoes not require baculovirus infection for its activity. The gp64promoter is transactivated by IE-1 but retains basal levels of activitywithout transactivation (Blissard, J. Virol. 65:5820-5827, 1991,Blissard, Virology. 190:783-793, 1992). The sequences responsible forIE-1 transactivation have been identified and are separable from thebasal promoter (Blissard, 1992). A long (500 bp upstream of the ATG) anda short version (100 bp upstream of the ATG) for each promoter wereobtained and cloned in place of the OpIE-1 promoter using TOPO-mediatedligation. LacZ was cloned into the resulting vectors. These constructstogether with the OpIE1 promoter version of pIB LacZ/V5-His weretransfected into Sf21 cells and polyclonal cultures were selected at twodifferent dosages of blasticidin. The longer gp64 construct apparentlydid not provide sufficient levels of bsd expression and the cells diedwith the control cells. Surviving stable cultures were obtained from theother four constructs. Cells were harvested after two weeks of selectionand expression levels were measured using β-galactosidase assays (FIG.23). β-galactosidase activities for stable cell cultures establishedwith different versions of pIB/V5-His. 20 μg of protein was used perassay. Higher levels of expression were obtained for all three alternatepromoters than obtained with the OpIE-1 promoter at both 20 and 100μg/ml blasticidin. There were not clear differences in LacZ activitybetween cultures selected at either concentration of blasticidin.

[0625] The gp64s promoter construct was used for GATEWAY™ adaptation. Toexamine the cloning efficiency and gene expression for the gp64sGATEWAY™ adapted version of this vector, four genes (Apolipoprotein,Calmodulin, TFIIs, and LacZ) were transferred into GATEWAY™ adaptedversions of pIB/V5-His and pIB/V5-His gp64 the vector using an LRreaction. All LR reactions resulted in thousands of colonies per μgplasmid and were correct when examined by agarose gel electrophoresis.Each construct was transfected into Sf21 cells. Transient and stableexpression of Apolipoprotein was compared between the gp64 and OPIE-1versions of pIB Apolipoprotein/V5-His GATEWAY™. Transient expressionlevels were equivalent between the gp64 and OpIE1 versions (FIG. 24,lanes 1 and 2), but expression was higher for the gp64 version followingselection (FIG. 24, lanes 3 and 4). To be sure that the higher stableexpression level observed for the gp64 promoter was a generalphenomenon, expression of Calmodulin, TFIIS, and LacZ between gp64 andOpIE-1 versions of pIB/V5-His GATEWAY™ were compared (FIG. 25). FIG. 25Ashows expression of calmodulin and TFIIs from Sf21 cells stablytransfected with OpIE-1 (lanes 1 and 3) and gp64s versions ofpIB/V5-His. 8.6 μg total protein was loaded per lane. FIG. 25B showsexpression of LacZ from Sf21 cells stably transfected with OpIE-1(lane 1) or gp64s (lane 2) versions of pIB/V5-His. Lane 3 is anon-transfected control. 5.7 μg of protein was loaded per lane. As forApolipoprotein, expression of Calmodulin, TFIIS (FIG. 25A) and LacZ(FIG. 25B) was higher from the gp64 version.

[0626] The above experiments were conducted with Sf21 cells in serumcontaining media. Use of a different promoter for expression of theantibiotic resistance marker could alter the dynamics of selection as afunction of cell type or media used. Selection and expression fromHighFive cells in serum- and serum free media was analyzed. In general,non-transfected cells were dead within a week but cells selected in SFMtended to die sooner (3-4 days) than those selected in media containingserum. As with the previous experiments, higher levels of geneexpression were obtained from the gp64 construct with stably transfectedHighFive cells, whether they were grown in serum or serum free media.Similar results were obtained with Sf21 cells in SFM media. FIG. 26shows High five cells grown in serum and serum free media transfectedwith Gp64 and OpIE-1 versions of pIB/V5-His. 24.5 μg total protein perassay.

[0627] A recombinational cloning adapted version of pIB/V5-His thatutilizes a different baculovirus promoter for expression of the bsd genehas been prepared. The basal gp64 promoter presumably results in lowerlevels of the bsd gene product than the OpIE-1 promoter used inpIB/V5-His and forces integration of the plasmid into more activechromosomal loci and/or at higher copy number.

Example 8

[0628] In some embodiments, the present invention provides a method ofmaking recombinant viruses using recombinational cloning. Onenon-limiting example is termed BaculoDirect™. Methods of this typeprovide a novel baculovirus cloning method that takes advantage ofrecombinational cloning technology (e.g., GATEWAY™ cloning technology,Invitrogen Corporation, Carlsbad, Calif.). With BaculoDirect™, an entryclone containing a nucleic acid sequence of interest (e.g., a sequencecomprising a gene of interest) may be recombined intorecombination-site-containing baculovirus genome in a one hour, in vitroreaction. The DNA product from this reaction can be transfected directlyinto suitable cells (e.g., Sf9 or Sf21 cells) to generate recombinantviruses and screen for expression. The ability to clone the sequence ofinterest (e.g., gene of interest (GOI)) directly into the baculovirusgenome in vitro contrasts with existing baculovirus cloning methods inwhich the recombination step is performed in insect cells or bacteria.Compared with these existing baculovirus technologies, BaculoDirect™ issignificantly faster, requires less hands-on time, and is more reliable.It is also easily adapted for high-throughput experiments. Thus,BaculoDirect™ offers significant advantages over current baculoviruscloning systems.

[0629] Throughout this disclosure, the term gene of interest (GOI) maybe used for the sake of convenience. This should not be construed aslimiting the present invention to nucleic acid sequences comprisinggenes. Any nucleic acid sequence of interest can be inserted into avector of the invention using materials and methods described herein.

Introduction

[0630] Baculoviruses are one of the most commonly used tools foreukaryotic expression of heterologous proteins. Traditionally, a GOI hadto be first cloned into a transfer vector and then moved into the virusby homologous recombination into the polyhedrin locus in permissiveinsect cells. This occurred at low frequency. Plaque assays were tediousand required identification of polyhedrin negative plaques from amongmuch more numerous polyhedrin-positive plaques.

[0631] During the last 20 years, innovations have made baculoviruscloning more convenient. Use of linearized DNA and design of therecombination strategy such that recombination restored function of anessential baculovirus gene boosted the proportion of recombinant plaquesobtained from 1-2% to over 90% (Kitts and Possee. 1993. BioTechniques14:810-817). However, multiple rounds of plaque purification were stillrequired and the entire process of obtaining a useful viral stock took3-4 weeks and a substantial amount of labor. Expression kits that usethis technology are marketed by BD Biosciences Pharmingen, San Diego,Calif. (Baculogold™), Novagen Inc., Madison, Wis. and InvitrogenCorporation, Carlsbad, Calif. (Bac-n-Blue™).

[0632] A second method for baculovirus cloning utilizes site-specificrecombination in bacteria to introduce the GOI into the baculovirus DNA(Luckow, et al., 1993. J. Virol. 67:4566-4579). The GOI is cloned into atransfer plasmid and used to transform a specialized bacterial strainthat contains the baculovirus genome propagated as an F′ plasmid(bacmid). The GOI is then introduced into the bacmid by site-specificrecombination between Tn7 sites on the transfer plasmid and in thebaculovirus genome. Bacteria containing recombinant bacmids are thenselected using antibiotic selection markers with appropriate selectivemedia. The bacmid DNA is extracted and then transfected into insectcells. Plaque purification is, in theory, not required (except for themost rigorous applications) and the entire process from transfer plasmidto pure virus stock requires 10-12 days. Invitrogen Corporation,Carlsbad, Calif. markets this system under the trade name Bac to Bac™,catalog number 10359-016.

[0633] While these advancements in baculovirus cloning have greatlysimplified use of baculovirus for routine protein expression, themethods described above still require significant “hands-on” time andare not well suited for parallel processing of multiple genes (i.e.,high-throughput). The present invention provides a new method thatgreatly simplifies and shortens the process for cloning and purificationof baculovirus recombinants. One non-limiting example of the presentinvention is BaculoDirect™, which utilizes GATEWAY™ recombinationalcloning technology (Invitrogen Corporation, Carlsbad, Calif.) torecombine a GOI into the baculovirus genome in vitro in a one hour, roomtemperature reaction. The resulting recombinant virus DNA is transfecteddirectly into insect cells. In just six days, cells can be harvested forexpression screening to obtain a pure viral supernatant suitable forcreation of high titer stocks.

Materials and Methods

[0634] All materials used in this study were from InvitrogenCorporation, Carlsbad, Calif. except restriction enzymes (Roche AppliedSciences, Indianapolis, Ind. or NEB, Beverly, Mass.) and ganciclovirsodium salt (GCV, Invivogen, San Diego, Calif. Catalog #sud-gcv).

[0635] Cells and Virus

[0636] Sf21 cells were cultured in Grace's medium with supplements and10% FBS unless stated otherwise. Infection of cells with wild typeAcMNPV or other viruses was performed as described (O'Reilly et al.,1992. Baculovirus Expression Vectors: a Laboratory Manual. W. H. FreemanCo., New York).

[0637] Plasmid and Virus Construction

[0638] Three versions of BaculoDirect™ were constructed. The firstcontained the melittin secretion signal, the second contained both amelittin signal and a C-term V5/His tag, and the third had a C-termV5/His tag without a secretion signal. FIGS. 19A and 19B provideschematics of recombination cassettes having a C-terminal V5/His tagwith (19B) and without (19A) a melittin leader.

[0639] The plasmid pVL1393 GST p10 stop (FIG. 34) was digested withBamHI and NcoI. A 15 kb band was purified (removing the GST tag) towhich was ligated, a double stranded oligonucleotide containing themelittin signal flanked by BamH1 and NcoI overhangs. The ligatedproducts were transformed into TOP10 bacteria and the correct clonesverified by restriction digestion and sequencing. This plasmid (pVL1393Mel Stop) contained a stop codon downstream of the attR2 site that hadto be removed by PCR directed site-specific mutagenesis. Primers EcoRIsense (GAA TTCCAGCTGAGCGCCGGTCGCTAC) and BglII antisense(AGATCTTCATTCATTCTCACCACTTTGTACAAG) were used to amplify a fragment frompVL1393 Mel Stop, and the resulting 209 bp fragment was cut with EcoRIand BglII, and then ligated to pVL1393 Mel Stop cut with the sameenzymes. The correct clone was identified by restriction digestion andsequence analysis. This gave pVL1393 Mel no-Stop.

[0640] Next, a V5-His tag was added downstream of the attR2 site. TheV5/His sequence was amplified from pIND/V5-His-TOPO (catalog no.K101001, Invitrogen Corporation, Carlsbad, Calif.) with primerscontaining BglII sites at each 5′ end (V5/His 5′:AGATCTGGGGAAGCCTATCCCTAACCC; V5/His 3′:AGATCTTCAATGGTGATGGTGATGATGACCGG). The amplicon was cloned into pCR2.1TOPO TA and then removed by BglII digestion and ligated to pVL1393 Melno-Stop cut with BglII. The correct clones were identified and verifiedby sequencing. This resulted in plasmid pVL1393 MeVV5-His. The melittinsignal was subsequently removed by replacing the melittin-attR1 sequencefrom pVL1393 Mel/V5-His with the attR1 sequence from pVL1393-Native,using NotI and BamHI. The correct plasmid clones were verified bysequencing and dubbed pVL1393 V5/His. FIG. 27 shows a schematic of thestrategy for construction of BaculoDirect™ DNA. In FIG. 27A, theGATEWAY™ counter selection cassette was cloned in the polyhedrin locusof wt AcMPNV by homologous recombination between with pVL1393 V5-His.The resulting virus DNA contains the counter selection cassette boundedby attR sites, immediately downstream of the polyhedrin promoter andupstream of the V5/His tag. In FIG. 27B, LR recombination betweenBaculoDirect™ DNA and an entry clone results in an expression virus inwhich the counter selection cassette is replaced by gene of interest.

[0641] Generation of BaculoDirect™ viruses

[0642] BaculoDirect™ viruses were created via conventional homologousrecombination between wt AcMNPV and homologous recombination sequencescontained in pVL1393 (FIG. 27, O'Reilly, et al., 1992). Briefly, Sf21cells were co-transfected with 0.5 μg wild type AcMNPV E2 virus DNA and3-5 μg of pVL1393 V5/His. After five days, the supernatant wascollected. This supernatant contained a mixture of recombinantBaculoDirect™ virus and wt virus. The recombinant virus was isolated andpurified through three to four rounds of plaque purification (O'Reilly,et al., 1992). Recombinant plaques could be distinguished from wt byphenotype, i.e., recombinant plaques were β-Gal⁺, polyhedra(−) whereaswt plaques were β-Gal(−), polyhedra(+).

Generation of Recombinant Expression Virus

[0643] Expression viruses were generated by performing standard LRclonase reactions between BaculoDirect™ DNA and entry clones containinga GOI flanked by attL1 and attL2 (FIG. 27B, GATEWAY™ Instruction ManualVersion C, 6/02, Invitrogen Corporation, Carlsbad, Calif.). Whereindicated, BaculoDirect™ DNA was linearized using AocI (an isoschizomerof Bsu36I), which cuts in the 5′ end of the lacZ gene. Reactions wereperformed with or without linearization. Twenty microliter LR reactionscontained 300 ng viral DNA, 100 ng entry clone, 4 μl LR clonase buffer,4 μl LR clonase, and were incubated for 1 h at room temperature. Twomillion Sf21 cells were transfected with varying amounts of completed LRreaction using 6 μl of Cellfectin® (catalog no. 10362-010, InvitrogenCorporation, Carlsbad, Calif.) and Sf900II media per the manufacturer'sinstructions. Five hours post-transfection, transfection buffer wasreplaced with the Grace's Supplemented Insect Medium containing 10% FBSand 100 μM ganciclovir. Three to five days later, the supernatant wascollected and varying amounts were used to infect fresh Sf21 cells withor without ganciclovir selection.

[0644] High Throughput (HTP) Screening of Expression

[0645] A method for performing LR reactions and transfection in 96 wellplate format was developed. FIG. 28 provides a schematic illustration ofBaculoDirect™ cloning and expression in 96 well plates. Entry vectorDNAs, diluted Cellfectin®, and Sf21 cells were arrayed in 96 wellplates. By arraying the components separately, the number of pipettingmanipulations of the Baculovirus DNA is minimized. Following expressionscreening from the first generation transfection, only those wellsshowing expression of a protein of interest need be processed further.

[0646] Three 96 well plates were needed in this experiment. In plate A,10 μl LR reactions were assembled in individual wells, starting withfive different entry plasmids arrayed in multiple wells. The entryclones used were: pENTR APO/V5-His (Apolipoprotein), pENTR CAL/V5-His(Calmodulin), pENTR GUS, pENTR LacZ and pENTR CAT. Each 10 μl reactionincluded 50 ng entry clone, 150 ng purified linear BaculoDirect™ DNA, 2μl LR clonase buffer, and 2 μl LR clonase. The LR reactions wereincubated in the plates for 1 h at RT. During the LR incubation, Sf21cells were seeded at 4.8×10⁴ cells per well in a separate plate andallowed to attach in plate B. In plate C, 2 μl of Cellfectin® werediluted to 40 μl per well with Grace's medium. After the 1 h LRreaction, 40 μl of Grace's unsupplemented media were added to each wellof plate A. Forty microliters of the Cellfectin® mixture from plate Cwere added to the diluted LR reactions and incubated at 27° C. for 30-45min. After this incubation, 150 μl of Grace's un-supplemented media wasadded to the wells of plate A. The cells in plate B were washed twice inGrace's media and then replaced with various amounts of the transfectionmixture from plate A. Plate B was incubated for 5 h at 27° C., and thenthe transfection mixture was removed and replaced with Grace's completemedia with 100 μM ganciclovir. The cells were allowed to grow for 3-4days. Supernatants from each well were transferred to a separate plate.The cells remaining in plate A were lysed in situ with 100 μl LDS lysisbuffer and heated to 80° C. for 5 min. Because apolipoprotein wassecreted, 15 μl of supernatant was denatured in 4× sample buffer.Protein samples were separated on SDS-PAGE gels, transferred to PVDF andvisualized by western blot.

Estimation of Viral Titers

[0647] Virus titers were estimated using two methods. Virus plaqueassays were performed using techniques well known in the art (e.g., Bacto Bac Baculovirus Expression System Manual, catalog no. 10359-016,version C, p. 27, Invitrogen Corporation, Carlsbad, Calif.). P1 or virussupernatants (infection from the P1 stock) usingapolipoprotein-expressing versions of each virus were serially dilutedten fold from 10⁻¹ to 10⁻⁸ and used to infect 2 million cells in sixwell plates. Recombinant plaques were counted and titers estimated basedon the dilution factor for each plate.

[0648] TCID₅₀ (Tissue Culture Infective Dose) measurements wereconducted as described (O'Reilly, et al., 1992). Briefly, a 96 wellplate was seeded with 4.8×10⁴ Sf21 cells per well. P1 stocks or virussupernatants were as described above. 10 μl of each dilution was addedper well, twelve wells per dilution, using a multi-channel pipettor. TheTCID₅₀ was calculated using the Excel (Microsoft) spreadsheet describedin O'Reilly, et al., 1992.

Results Optimization of LR Clonase Reactions Using Baculodirect™ DNA

[0649] BaculoDirect™ DNA is the functional equivalent of a GATEWAY™destination vector. GATEWAY™ destination vectors designed for use inbacteria, e.g., E. coli, contain a counter-selection cassette containingthe ccdB gene and a chloramphenicol resistance marker, bounded by attRsites. Recombination between an attL containing entry clone and thedestination plasmid replaces the ccdB gene and Chl(r) marker with thegene of interest, yielding an expression clone bounded by attB sites.This selection scheme does not work in insect cells. To create acounter-selection cassette for use with baculovirus, wild typebaculovirus DNA was engineered with a cassette containing the herpesvirus TK gene (HSV tk) and the lacZ gene, both under control ofbaculovirus promoters, bounded by attR sites (FIG. 27A). The attRcassette was placed immediately downstream of the polyhedrin promoter.Recombination between the “destination virus” and an entry clonereplaces the counter selection cassette with the GOI under polyhedrinpromoter control (FIG. 27B). Transfection of the resulting DNA creates amixed baculovirus infection with both recombinant virus and parent viruspresent. Replication of the parent virus is prevented by growing thecells in the presence of ganciclovir, which is metabolized by the HSV tkgene into a toxic inhibitor of DNA replication (Godeau, et al., 1992,Nucl. Acids Res. 20:6239-6246). Cells that are infected by parent viruswill also express the lacZ gene, which can be assayed by staininginfected cells, providing a method for checking the purity the virusinfection.

[0650] To test if the LR reaction would work between a 3-4 kb entryclone and the 140 kb BaculoDirect™ virus DNA, an LR reaction betweenmelittin BaculoDirect™ and a GFP entry clone was performed. GFPexpression was clearly visible by fluorescence as early as 48 hpost-transfection and was stronger at 72 h, demonstrating that the LRreactions were successful and that GFP was placed under control of thepolyhedrin promoter. Transfection, infection and selection conditionswere then optimized to minimize background resulting from residualparental virus, as evidenced by GFP fluorescence and β-galactosidasestaining.

[0651] Linear and circular BaculoDirect™ DNA were compared. Thus, astandard LR reaction was performed with either linearized or circular(uncut) melittin BaculoDirect™ DNA, without ganciclovir selection. Ten,twenty or thirty microliters of LR reaction were used to transfect Sf21cells (only the results from the 20 μl transfection are shown in FIG.29). Three days later, varying amounts of supernatant (P1 stock from the“first generation”) from each transfection were used to infect new Sf21cells. After four days, infected cells were examined for GFPfluorescence and then stained for β-galactosidase activity. These cellsare from the “second generation” and the supernatant from them is asmall scale high titer stock (see titer data below). Virtually all cellsin all treatments were fluorescent, demonstrating that a productivebaculovirus infection had been established and that the virus wasactively expressing GFP. FIG. 29 shows the results of an analysis ofcells transfected with LR reaction products from the melittin version ofBaculoDirect™ DNA. LR reactions between melittin BaculoDirect™ DNA wereperformed with AocI cut or circular virus DNA and a GFP entry clone.Sf21 cells were transfected with 10 μl, 20 μl or 30 μl each LR reaction,using either linear virus DNA or circular virus DNA as indicated,without GCV selection. Cells were examined by fluorescence and β-Galstaining 72 hours following transfection. The result here shown from the20 μl of LR reaction was typical. Some β-Gal positive cells were foundin every well examined. β-galactosidase activity (i.e., background) wasmuch higher in cells that had been infected with P1 virus derived fromcells transfected with LR reactions that used circular rather thanlinearized BaculoDirect™ DNA (FIG. 29, upper panel). Background was muchlower if cells were infected with P1 stocks derived from LR reactionswith linearized BaculoDirect™ DNA, although some background was detectedwhen more P1 stock was used for infection (FIG. 29, lower panel).

[0652] The effect of ganciclovir selection was then tested by growingcells in the presence or absence of ganciclovir. LR reactions wereperformed as above with circular or linearized melittin BaculoDirect™DNA. SF21 cells were transfected with varying amounts of LR reaction andthen grown without GCV (first generation). After 72 h, varying amountsP1 stock obtained from each transfection were used to infect new Sf21cells, now grown in the presence of 100 μM GCV. After 4 days (secondgeneration), the cells were examined for GFP fluorescence and stainedfor β-galactosidase. GCV did not appreciably reduce the number of cellsstaining positive for β-galactosidase activity when infections werederived from LR reactions using circular virus, whereas GCV reduced thenumber of β-gal positive cells from infections derived from LR reactionsthat used linearized virus.

[0653] The effectiveness of ganciclovir in eliminating background whenused during the first generation, the second generation or bothgenerations was tested. When ganciclovir was used in the firstgeneration or the second generation, at least some blue cells wereobserved following the second generation. In general, more backgroundwas found when more LR reaction was transfected. However, whenganciclovir was used in both generations, no blue cells were found,suggesting that there were no cells infected by parent virus followingtwo rounds of ganciclovir selection. Moreover, zero background was foundirrespective of how much LR reaction was used during for thetransfection. Representative results from these experiments are shown inFIG. 30. In the experiment shown in FIG. 30, cells were transfected andselected during both generations as described above. Following thesecond generation, the cells were photographed to illustrate typicalresults following the selection protocol. Essentially all cells wereproducing GFP, but no cells stained positive for β-Gal if GCV selectionwas maintained during both generations. Thus, parent virus is notreplicating in these cells. These results were obtained with cells grownin serum. The same result was found when serum free-adapted Sf21 cellswere grown and selected with GCV in serum free media. Similar resultswere subsequently found using linearized V5/His virus.

High-Throughput Screening of Expression

[0654] The ability to clone and express genes from baculovirus withoutplaque purification or selection in bacteria suggests that BaculoDirect™can be used conveniently for high-throughput screening of expression.Five pENTR clones were chosen (CAT, GUS, LacZ, Apolipoprotein/V5-His,and Calmodulin/V5-His) for expression. Each pENTR DNA was arrayed inmultiple wells of a 96 well plate as illustrated in FIG. 28. LR clonasereaction mixes were added as described in the Materials and Methods,using linearized V5/His BaculoDirect™ DNA. All manipulations usedmulti-channel or repeating pipettors and thus could also be performedrobotically. Following ganciclovir selection during the firstgeneration, expression from each virus was assayed by western blot. Allfive genes expressed at levels sufficient to be easily detected (FIG.31). The supernatants were stored in a separate 96 well plate and wereavailable for second round infection and selection.

[0655]FIG. 31 shows the results of the screening of protein expressionfrom LR reactions performed in a 96 well plate. Indicated pENTR DNAswere arrayed in a 96 well plate, and LR reactions were performed asdescribed above. Supernatants were removed to a separate plate and thencells were lysed using 100 μl LDS sample buffer. 15 μl of lysate wasapplied per lane except for apolipoprotein, which was secreted. Forapolipoprotein, 11 μl of supernatant was used instead of cell lysate.The blot was visualized with anti V5:AP conjugate at 1:5000 and exposedto film for 15 sec.

[0656] Titer Comparison

[0657] Two methods were used to compare the virus titers obtained fromBaculoDirect™ with Bac to Bac™ or Bac-n-blue™. Apolipoprotein was clonedinto pBlueBac 4.5/V5-His and co-transfected this with linear Bac-n-BlueDNA into Sf21 cells using well known techniques (e.g., Bac-n-blue™manual, catalog no. K855-01, version M, Invitrogen Corporation,Carlsbad, Calif.). Following one round of plaque purification, a hightiter stock was made. The entire apolipoprotein/V5-His reading frame wascloned into pFastBac, and bacmid DNA was generated using standardtechniques (e.g., Bac to Bac™ manual, catalog nos. 11827-011, 11806-015,11804-010 and 11807-03, version C, Invitrogen Corporation, Carlsbad,Calif.). The bacmid DNA was transfected into Sf21 cells, and a hightiter stock was made. The apolipoprotein/V5-His reading frame was alsocloned into pENTR and transferred in an LR reaction into linearizedV5-His BaculoDirect™ DNA. Titers were measured following transfectionand infection using plaque assay and TCID₅₀ methods. The titers obtainedfollowing infection were similar for all three baculovirus expressionsystems using either method and were in the range of 3×10⁸ to 7×10⁸pfU/ml (FIG. 32). 10⁸ pfu/ml is a typical titer for baculovirus and thusBaculoDirect™ baculoviruses replicate as well as the baculoviruses usedin other systems.

[0658]FIG. 32 shows an estimation of virus titers using plaquepurification and TCID₅₀ measurements. Apolipoprotein was cloned intopENTR, pFASTBAC, or pBlueBac 4.5/V5-His (catalog no. V207520, InvitrogenCorporation, Carlsbad, Calif.). Procedures for MaxBac and Bac to Bacwere followed as described in their respective instruction manuals.Dilutions of P1 or virus stock from second generation supernatants wereserially diluted and used to infect cells for agar overlay (plaquepurification) or in 96 well plates (TCID₅₀). For BaculoDirect™, cellswere selected on 100 μM ganciclovir for both generations. Titers werecalculated as described (O'Reilly, et al., 1992).

Discussion

[0659] BaculoDirect™ is functionally a GATEWAY™ adaptation of thebaculovirus genome. Lambda-based recombination occurs between the attRsites engineered in the baculovirus genome and attL sites surroundingthe GOI in an entry clone. Following the LR clonase reaction, thecounter-selection cassette containing the HSV tk gene and lacZ driven bybaculovirus promoters and bounded on each side by attR sites on thebaculovirus is replaced by the GOI from the entry clone. This results inre-circularization of the virus DNA. Replication of parent virus isprevented, both because it remains linearized, and because the tk geneproduct prevents DNA replication in the presence of ganciclovir.Linearization was highly effective at preventing replication of parentalvirus (FIG. 29). Virtually all cells expressed β-Gal followingtransfection and ganciclovir selection if the LR reaction was performedwith circular virus, whereas use of linear virus boosted to greater than95% (FIG. 29).

[0660] The presence of lacZ in the counter-selection cassette provides ameans of judging the purity of virus stocks, since the absence of β-Galcell staining is a good indication that a virus stock is free ofcontaminating parent virus.

[0661] Depending on the context, the attB2 site can sometimes pose aproblem for expression and or detection from the C-terminal V5 epitopetag. In FIG. 31, APO and CAL were cloned without an internal attB2 site,while the remaining three genes were cloned with an internal attB2 sitebetween the gene and the V5-His tag. The entry clones used for APO andCAL had an encoded C-terminal V5-His. All of the genes except for CALappeared to be expressed and detected at high levels (FIG. 31). It hasbeen observed that CAL tends express at lower levels in mostexperiments. BaculoDirect™ viruses that express GUS with or withoutattB2 inside the reading frame have been constructed. GUS expression wasdetected equally well for both versions, suggesting that the attB2 sitedoes not appear to interfere with expression or detection from the V5tag in the context in which it is used in BaculoDirect™.

[0662] The addition of the GATEWAY™ cassette, presence of attB sites inthe polyhedrin locus, or ganciclovir selection, did not appear to affectvirus titers when compared to other baculovirus expression systems (FIG.32). Titers in excess of 10⁸ pfu/ml were obtained routinely followingthe second-generation virus infection. Since the virus stocks obtainedfollowing selection on ganciclovir were found to be essentially pure(based on the lack of infected cells that were β-Gal positive), thevirus supernatants obtained at this stage require no plaque purificationand can be scaled up for production of high titer stocks. The entireprocess, from LR cloning to pure virus stock can be performed formultiple genes simultaneously in 96 well plates. Although the presentmethodology has been exemplified using just five genes, one of skill inthe art will appreciate that any number of genes (e.g., 20, 50, 100,250, 500, 1,000, 2,500, 5,000, etc.) can be processed in a similarmanner. The present method allows screening for expression after justthree days, and continued selection and scale-up can focus on only thosewells that express the desired protein product.

[0663]FIG. 33 shows a comparison of the time required for expressiontesting and virus purification between BaculoDirect™ and Bac to Bac.Numbers next to the arrows between steps are cumulative labor time inhours. Chronological elapsed times are indicated in days. Procedurescommon to both systems were given equal times, e.g., 2 hours fortransfection, 4 hours for expression testing.

[0664] Compared to other baculovirus expression systems, the methodsdescribed herein (e.g., BaculoDirect™) require much less hands-on timeand are faster chronologically. For example, Bac to Bac™ requires 10days to obtain a purified viral stock and upwards of 17 hours of actuallabor (FIG. 33) This assumes that the P1 stock obtained with Bac to Bac™does not require plaque purification. In practice, one of skill in theart is likely to have difficulty in obtaining pure stock without plaquepurification; as a result, plaque purification is now being encouragedfor Bac to Bac™ users. The MaxBac baculovirus expression system relieson homologous recombination in insect cells, and, like other methodsutilizing homologous recombination, requires plaque purification andeven more chronological time and labor. By contrast, BaculoDirectrequires only 8 hours of labor over six days to obtain a purified virusstock suitable for production of high titer stocks.

[0665] In summary, one of skill in the art with a collection of clonesadapted for use in recombinational cloning methods (e.g., pENTR clonesadapted for GATEWAY™ methods) will be able to clone and express theirgenes of interest quickly in a baculoviral expression system of thepresent invention, using simple protocols, and in parallel reactions.

[0666] A suitable protocol for production of the recombinantbaculoviruses of the invention is as follows:

[0667] Materials: Sf9 or Sf21 cells growing in log phase; linearizedBaculoDirect Virus DNA; GOI cloned into L1/L2 Entry clone (e.g., pENTRCAT; LR clonase Buffer; LR clonase; and ganciclovir sodium (100 mMsolution in water).

[0668] A sequence of interest may be cloned into L1/L2 entry vector.Suitable cells (e.g., Sf9 or Sf21 cells) may be plated at recommendeddensities (e.g., Guide to Baculovirus Expression Systems and Insect CellCulture, catalog nos. 10359016, 10360014, 10608016, 11827011, InvitrogenCorporation, Carlsbad, Calif., Feb. 27, 2002). HighFives are lesspreferred as they give low infectivity/titer. A suitable method mayemploy 6 well plates with 2 million Sf21 cells. An LR reaction may beperformed between Entry vector and BaculoDirect™ linearized DNA(GATEWAY™ Manual) using 100 ng entry vector and 300 ng linearizedBaculoDirect™ DNA. 1 h at room temperature. An aliquot (e.g., 10 μl) ofLR reaction may be transfected into the cells (e.g., using Cellfectin®protocol). Transfection media may be replaced with growth media ofchoice, supplemented with 100 μM ganciclovir. After 72 hours, an aliquot(e.g., 10 μl) of supernatant from transfected cells can be added tofresh well of cells with 100 μM ganciclovir in growth medium. Proteinexpression can be checked by western blot at this time. After 72 hours,supernatant can be collected (e.g., in a sterile tube)x. Recommended:Stain cells with β-Gal staining kit. Viruses may be amplified as perstandard protocols.

Example 9

[0669] In some embodiments, the present invention provides materials andmethods for the construction and use of recombinant retroviruses, e.g.,lentiviruses. Although the present invention is exemplified using alentivirus, any other type of retrovirus may be used in an analogousfashion to practice the present invention. A commercially availablesystem for the construction of recombinant lentiviruses is ViraPower™Lentiviral Expression System, available from Invitrogen Corporation,Carlsbad, Calif. The ViraPower™ system provides a retroviral system forhigh-level expression in dividing and non-dividing eukaryotic cells,e.g., mammalian cells. Examples of products available from InvitrogenCorporation, Carlsbad, Calif. include the ViraPower™ LentiviralDirectional TOPO® Expression Kit catalog number K4950-00, the ViraPower™Lentiviral GATEWAY™ Expression Kit catalog number K4960-00, and theViraPower™ Lentiviral Support Kit catalog number K4970-00.

[0670] The present invention permits one skilled in the art to createreplication-incompetent lentiviruses to deliver and express one or moresequences of interest (e.g., genes). These viruses (based loosely onHIV-1) can effectively transduce dividing and non-dividing mammaliancells (in culture or in vivo), thus broadening the possible applicationsbeyond those of traditional Moloney (MLV)-based retroviral systems(Clontech, Stratagene, etc.). Directional TOPO and GATEWAY™ lentiviralvectors have been created to clone one or more genes of interest with aV5 epitope, if desired. The vectors also carry the blasticidinresistance gene (bsd) to allow for the selection of transduced cells.Without additional modifications, these vectors can theoreticallyaccommodate up to ˜6 kb of foreign gene. Three supercoiled packagingplasmids (gag/pol, rev and VSV-G envelope) are provided to supply helperfunctions and viral proteins in trans. Finally, an optimized producercell line (293FT) is provided that will facilitate production of hightiter virus. A schematic representation of the production of a nucleicacid molecule comprising all or a portion of a lentiviral genome isshown in FIG. 35. Plasmid maps of vectors adapted for use with GATEWAY™and topoisomerase cloning in the production of nucleic acid moleculescomprising all or a portion of a lentiviral genome are shown in FIGS.36A (pLenti6/V5-DEST), 36B (pLenti6/V5-D-TOPO®), 36C (pLenti4/V5-DEST),and 36D (pLenti6/UbC/V5-DEST) respectively. The nucleotide sequences ofthe plasmids are provided in Tables 17-20. Plasmid maps of the threepackaging plasmids pLP1, pLP2, and pLP/VSVG are shown in FIGS. 37A, 37B,and 37C respectively and the nucleotide sequences of these plasmids areprovided as Tables 21, 22, and 23, respectively.

[0671] Retroviruses are RNA viruses that reverse transcribe their genomeand integrate the DNA copy into a chromosome of the target cell. It wasdiscovered that the retroviral packaging proteins (gag, pol and env)could be supplied in trans, thus allowing the creation of replicationincompetent viral particles capable of stably delivering a gene ofinterest. These retroviral vectors have been available for gene deliveryfor many years (Miller et al., (1989) BioTechniques 7:980-990). Onesignificant advantage of retroviral-based delivery is that the gene ofinterest is stably integrated into the genome of the host cell with veryhigh efficiency. In addition, no viral genes are expressed in theserecombinant vectors making them safe to use both in vitro and in vivo.However, one main drawback to the traditional Moloney-based retrovirusesis that the target cell must undergo one round of cell division fornuclear import and stable integration to occur. Traditional retrovirusesdo not have an active mechanism of nuclear import and therefore mustwait for the host cell nuclear membrane to breakdown during mitosisbefore they can access the host genomic DNA (Miller et al., (1990) Mol.Cell. Biol. 10:4239-42).

[0672] Unlike traditional retroviruses, HIV (classified as a“lentivirus”) is actively imported into the nuclei of non-dividing cells(Lewis et al., (1994) J. Virol. 68:510-516). HIV still goes through thebasic retrovirus lifecycle (RNA genome reverse transcribed in the targetcell and integrated into the host genome); however, cis-acting elementsfacilitate active nuclear import, allowing HIV to stably infectnon-dividing cells (for reviews see Buchschacher et al., (2000) Blood95:2499-2504, Naldini et al., (1999) “The Development of Human GeneTherapy”, Cold Spring Harbor Laboratory Press, pages 47-60). It isimportant to note that, for both lentivirus and traditionalretroviruses, no gene expression occurs until after the viral RNA genomehas been reverse transcribed and integrated into the host genome.

[0673] Similar to other retrovirus expression systems, the packagingfunctions of HIV can be supplied in trans, allowing the creation oflentiviral vectors for gene delivery. With all the viral proteinsremoved, the gene delivery vector becomes safe to use and allows foreignDNA to be efficiently packaged. In addition, it has been shown thatlentiviral (or any retroviral) envelope proteins can be substituted forones with broader tropism. The substitution of envelope is calledpseudotyping, and allows creation of lentiviral vectors capable ofinfecting a wider variety of cells besides just CD4+ cells. Many havefound that the G protein from vesicular stomatitis virus (VSV-G) is anexcellent pseudotyping envelope protein that imparts a very broad hostrange for the virus (Yee et al., (1994) Proc. Natl. Acad. Sci. USA91:9564-9568). The ability of pseudo-typed lentivirus to infect a broadrange of non-dividing cells has led to its extensive use in animal genedelivery and gene therapy (Baek et al., (2001) Hum Gene Ther 12:1551-8,Park et al., (2001) Mol Ther 4:164-73, Peng et al., (2001) Gene Ther8:1456-63).

Materials and Methods

[0674] Vector constructions. Lentiviral vector materials were receivedfrom Cell Genesys (Foster City, Calif., see U.S. Pat. Nos. 5,686,279;5,834,256; 5,858,740; 5,994,136; 6,013,516; 6,051,427; 6,165,782 and6,218,187, and Dull et al. (1998) J. Virol. 72(11):8463-8471) andmodified to incorporate a blasticidin expression cassette and the V5epitope tag using standard techniques to create pRRL6/V5 also referredto as pLenti6/V5. The nucleotide sequence of pRRL6/V5 is provided inTable 36. To create the GATEWAY™ Destination vector, pLenti6/V5-DEST,the Destination Vector Conversion cassette B (available from InvitrogenCorporation, Carlsbad, Calif. catalog #11828-019) was ligated intopRRL6/V5. This Destination vectorwas propagated in DB3.1 bacteria in thepresence of ampicillin (100 μg/ml) and chloramphenicol (15 μg/ml) tomaintain integrity. In one alternative of this aspect of the invention,the chloramphenicol resistance gene in the cassette can be replaced by aspectinomycin resistance gene (see Hollingshead et al., Plasmid13(1):17-30 (1985), NCBI accession no. X02340 M10241), and thedestination vector containing attP sites flanking the ccdB andspectinomycin resistance genes can be selected onampicillin/spectinomycin-containing media. It has recently been foundthat the use of spectinomycin selection instead of chloramphenicolselection results in an increase in the number of colonies obtained onselection plates, indicating that use of the spectinomycin resistancegene may lead to an increased efficiency of cloning from that observedusing cassettes containing the chloramphenicol resistance gene.

[0675] To create the control Moloney retroviral vector, prKAT6/V5-DEST,prKAT (Cell Genesys) was digested with BamHI and filled-in with Klenow.This was ligated to the 2732 bp fragment, containing the DEST cassetteand SV40-Bsd^(R) cassette, resulting from the digestion ofpLenti6/V5-DEST with SpeI and Acc65I followed by Klenow fill-in and gelpurification. This Destination vector was propagated in DB3.1 bacteriain the presence of ampicillin (100 μg/ml) and chloramphenicol (15 μg/ml)to maintain integrity. In one alternative of this aspect of theinvention, the chloramphenicol resistance gene in the cassette can bereplaced by a spectinomycin resistance gene (see Hollingshead et al.,Plasmid 13(1):17-30 (1985), NCBI accession no. X02340 M10241), and thedestination vector containing attP sites flanking the ccdB andspectinomycin resistance genes can be selected onampicillin/spectinomycin-containing media. It has recently been foundthat the use of spectinomycin selection instead of chloramphenicolselection results in an increase in the number of colonies obtained onselection plates, indicating that use of the spectinomycin resistancegene may lead to an increased efficiency of cloning from that observedusing cassettes containing the chloramphenicol resistance gene.

[0676] To create the expression control vector, pLenti6/V5-GW/lacZ, andthe cognate Moloney retroviral control vector, prKAT/V5-GW/lacZ,GATEWAY™ LR reactions were performed with each of the DEST vectors andan entry vector having a copy of the lacZ gene with no stop codonaccording to the manufacturer's protocol.

[0677] Directional TOPO adaptation. The pRRL6/V5 vector was propagatedin ampicillin (100 μg/ml) and blasticidin (10 μg/ml) to maintainintegrity and reduce backgrounds in the TOPO adaptation. The pRRL6/V5vector was Directionally TOPO-adapted at the EcoRI (5′ end) and XhoI(3′-end) sites. EcoRI buffer (New England Biolabs, Beverly, Mass.) wasused in the digest throughout; vectors were digested first for 3 hourswith XhoI at 6 units of enzyme/μg of DNA followed by a 3 hour digestionwith EcoRI at 4 units of enzyme/μg DNA. Digested DNA was purified byPhenol/Chloroform/Isoamyl alcohol (PCA) extraction, Ethanolprecipitation, 80% Ethanol wash, followed by isopropanol precipitationand another 80% ethanol wash to remove the enzymes and the ˜30 bpmulticloning site between the EcoRI and XhoI sites. At this point, theconcentration of the cut DNA was quantitated and 10 ng was transformedinto chemically competent TOP 10 E. coli to assess the amount of uncutvector (vector that had recombined to delete the multicloning site, orthe original vector which “evaded” both restriction enzymes activity).

[0678] The oligonucleotides used for directional adaptation are listedbelow:

[0679] EcoRI (5′ end): Non-Regenerative Site Topo-D15′ P-AATTGATCCCTTCACCGACATAGTACAG 3′ Topo-D2 5′ P-GGTGAAGGGATC 3′

[0680] XhoI (3′ end): Regenerative Site Topo-D65′ P-TCGAGCCCTTGACATAGTACAG 3′ Topo-D7* 5′ P-AAGGGC 3′

[0681] The oligonucleotides were used as pairs: Topo-D1/D2 andTopo-D6/D7 in 200 fold molar excess to vector (51 μg of Topo-D1/D2 pairand 40 μg of Topo-D6/D7 per 100 μg vector DNA). Topo-D1 and D2 werepaired in 2.3 to 1 mass ratio, respectively. Topo-D6 and D7 were pairedin 3.7 to 1 mass ratio, respectively.

[0682] 50 units of T4 DNA Ligase (New England Biolabs, Beverly, Mass.)per 1 μg of vector DNA was used in an overnight ligation (˜16 hours) ina 14° C. water bath to ligate the adapter oligonucleotides to thevectors. Subsequently, the sample was heated at 67.5° C. for 15 minutesand then re-digested with EcoRI at 2 units of enzyme/μg vector DNA for1.5 hours.

[0683] Free oligonucleotides were purified away from theoligonucleotide-adapted vector by PCA extraction and a Modified S.N.A.P.column purification protocol, as follows: The PCA extracted DNA (topaqueous phase) was added to 5 volumes of Modified Binding Buffer (MBB)[60% of S.N.A.P. Binding buffer: 40% of (100%) isopropanol], mixed andloaded onto a S.N.A.P. mini or midi (B) column; and the flow through wasreloaded back onto the column once more. The column was then washedtwice with SNAP Wash buffer, once with the Final Wash buffer (EtOH) andeluted in TE (60-100 μl for mini column and 750 μl for midi column) andconcentration determined spectrophotometrically (OD_(260/280)) producingpLenti6/V5-D-TOPO™.

[0684] At least 50 μg of the oligo adapted vector was “Charged” withvaccinia topoisomerase in the following reactions (reagents added in theorder listed):

[0685] Topo Charging with Kinase Volume Reagent Final Concentration # μlTopo adapted & purified DNA (at least 50 μg) # μl Topo D-70 annealing0.2 μg/μg vector DNA oligo 50 μl Vaccinia Topo Enzyme 1 μg Topo/μgvector DNA (1 mg/ml) # μl Water 5.3 μl 1 M Tris pH 7.5 15 mM 350 μlTotal

[0686] Incubate the reaction at 37 degrees Celsius water bath for 10minutes and then add: Volume Reagent Final Concentration 16.5 μl 100mMATP 1.35 mM ATP (33 mM ATP/μg DNA) 4 μl 1 M _(MgCl2) 10 mM MgCl₂ 33 μl10 Units/μl LTI 6.6 Units/ T4 DNA Kinase 1 μg DNA 403.5 μl Total

[0687] Incubate the reaction at 37 degrees Celsius water bath for 5minutes and then load all of reaction into Q-column.

[0688] TOPO Vector Purification. Q-column purification was performed onthe TOPO-charged sample with a 0-1M NaCl (50 mM Tris pH 7.5) gradient asreported for the TOPO-Adapted Entry vectors. DNA fluorescencecharacterization in the presence of Hoechst dye number 33258 (Sigmacatalog #B-2883) was used to quantitate the concentration of individualor pooled fractions containing column purified TOPO-charged vector. Ingeneral, approximately 50% of the total DNA loaded onto the column islost during the purification and the vector-TOPO complexes are eluted in˜500 mM NaCl. An equal volume of 2× TOPO-Vector Buffer (50 mM Tris 7.5,2 mM EDTA, 2.5 mM DTT, 0.1 mg/ml BSA, 0.1% Triton X 100, 90% glycerol)is added to the sample fractions. Therefore, the final TOPO VectorBuffer=50 mM Tris 7.5, 1 mM EDTA, 1.25 mM DTT, 0.05 mg/ml BSA, 0.05%TritonX-100, 45% Glycerol. Samples are stored at −20 degrees Celsiusuntil tested.

[0689] Standard Topogation reactions were set-up as follows:

[0690] 1 μl Topo-charged vector

[0691] 1 μl Directional insert PCR product*

[0692] 1 μl Salt Solution or 1 μl water

[0693] 3 μl water *Depending on the concentration of Topo-chargedvector, PCR product insert should be adjusted to maximize yield. Ratioof 1 ng vector: 1-2 ng 750 bp insert (Or 1:10-20, vector:insert molarratio) give good yields.

[0694] The topogation reactions were incubated at room temperature for 5min. Two microliters of the reaction was added to TOP 10 cells,incubated on ice for ˜20 min, heat shocked for 40 seconds at 42° C.,placed on ice, and then 250 μl of SOC was added to the transformedcells. Cells were shaken at 37° C. for 1 hr and 100 μl of the cellmixture was plated on LB-amp plates containing blasticidin (50 μg/mlfinal).

[0695] Cell culture and growth arrest. 293FT producer cells (availablefrom Invitrogen Corporation, Carlsbad, Calif., catalog number R7007)were cultured in DMEM/10% FBS/L-glutamine/non-essential aminoacids/penicillin/streptomycin containing 500 μg/ml G418. MJ90 primaryhuman foreskin fibroblasts, HT1080 human fibrosarcoma (ATCC #CCL-121)and HeLa cervical carcinoma cells (ATCC #CCL-2) were cultured inDMEM/10% FBS/non-essential amino acids/penicillin/streptomycin. Chinesehamster ovary cells (CHO-K1, ATCC #CCL-61) were cultured in Hams F12/10%FBS/L-glutamine/penicillin/streptomycin. For blasticidin selections, thefollowing final concentrations were used: HT1080: 10 μg/ml, CHO: 5μg/ml, HeLa: 2 μg/ml.

[0696] MJ90 primary cells were growth arrested by contact inhibition.Briefly, 1×10⁵ cells were plated per well of a 6-well plate and mediachanges were performed every 3 days for 7 to 14 days, or until aquiescent monolayer was achieved. Aphidicolin (Sigma, St. Louis, Mo.,catalog number #A0781) was used to arrest HT1080 cells at the G1/Stransition. Exponentially growing cultures were plated at 2×10⁵ cellsper well of a 6-well plate and the following day fresh media wassupplied containing 1 μg/ml aphidicolin. Transductions ofaphidicolin-arrested cells were performed in the continued presence ofdrug.

[0697] Primary, post-mitotic rat hippocampal and cortical neuronaltissues were received from BrainBits Inc. (Dr. Greg Brewer, Universityof Southern Illinois). Tissues were dissociated with a Pasteur pipette,spun down at 1100 rpm for one minute and resuspended in NeuroBasalMedium (Invitrogen Corporation, Carlsbad, Calif., Gibco #21103-049)containing B27 supplement (Invitrogen Corporation, Carlsbad, Calif.,Gibco #17504-010), 0.5 mM L-glutamine and 25 μM glutamate. 5×10⁴hippocampal or 1×10⁵ cortical neurons were plated per well in 24-wellplates. Four days after plating, half of the medium was removed andreplaced with complete NeuroBasal Medium (as above) but without theglutamate. The following day, cells were transduced with virus.

[0698] Virus production. For optimal virus production, 5×10⁶ 293FT cellswere plated per 100 mm plate. Twenty-four hours later, the culturemedium was replaced with 5 ml OptiMem/10% FBS (Opti-MEM®, catalog no.22600050, Invitrogen Corporation, Carlsbad, Calif.) and cells werequadruple co-transfected, as follows. 12 μg DNA total, at a mass ratioof 1:1:1:1 pLenti6/V5/gene:pLP-1:pLP-2:pLP/VSVG (3 μg of each DNA) wasmixed with 1.5 ml of OptiMem media. In a separate tube, 36 μl ofLipofectamine 2000 was also mixed with 1.5 ml of OptiMem media. After a5-minute incubation period at room temperature, the two mixtures werecombined and incubated at room temperature for an additional 20 minutes.At the completion of the incubation period, the transfection mixture wasadded to the cells dropwise and the culture plate was gently swirled tomix. The following day the transfection complex was replaced withcomplete media (DMEM, 10% FBS, 1% penicillin/streptomycin, L-glutamineand non-essential amino acids). Forty-eight to seventy-two hours posttransfection, the virus-containing supernatants were harvested,centrifuged at 3000 rpm for 15 minutes to remove dead cells and placedin cryovials in 1 ml aliquots. Titers were performed on freshsupernatants (see below) and the remaining viral aliquots were stored at−80° C.

[0699] Viral titering and transduction. All applications of virus tocells were performed in the presence of 6 μg/ml polybrene (Sigma, St.Louis, Mo., catalog #H9268) and media changes were performed 12-24 hourspost transduction. For titering virus, 6-well plates were seeded at2×10⁵ cells per well with HT1080 cells the day before transduction. Onewell served as an untransduced control (mock) and the remaining fivewells contained 1 ml each of ten-fold serial dilutions of viralsupernatant ranging from 10⁻² to 10⁻⁶ (see example below). The dilutionswere mixed by gentle inversion (dilutions should not be vortexed) priorto adding to cells. 6 μg/ml of polybrene was added to each well. Theplate was gently swirled to mix. The following day, the media wasreplaced with complete media. Forty-eight hours post transduction, thecells were placed under 10 μg/ml blasticidin selection (Invitrogen).After 10 to 12 days of selection, the resulting colonies were stainedwith crystal violet: A 1% crystal violet solution was prepared in 10%ethanol. Each well was washed with 2 ml PBS followed by 1 ml of crystalviolet solution for 10 minutes at room temperature. Excess stain wasremoved by two 2 ml PBS washes and colonies visible to the naked eyewere counted to determine the viral titer of the original supernatants.In a typical example, colonies can be counted in the 10⁻⁵ and 10⁻⁶dilutions.

[0700] Protein analysis. Total cell lysates were prepared using NP-40lysis buffer (Igepal CA636, Sigma, St. Louis, Mo.) and the proteins (20μg/lane) were separated on a 4-20% Novex Tris-Glycine gel. Followingelectrophoresis, the proteins were transferred to nitrocellulose.Western blotting was performed using the Western BreezeChemiluminescence Kit (Invitrogen Corporation, Carlsbad, Calif.), usinganti-large T antigen mouse monoclonal antibody (e.g., catalog no.554149, BD Biosciences Pharmingen, San Diego, Calif.), anti-lacZ rabbitpolyclonal antibody (1:5000 dilution, Invitrogen Corporation, Carlsbad,Calif.) or anti-V5 mouse monoclonal antibody (1:2000 dilution,Invitrogen Corporation, Carlsbad, Calif.). Beta-galactosidase activityassays were performed using the Galacto-Light Plus Kit (Tropix, AppliedBiosystems, Foster City, Calif.) according to the manufacturer'sinstructions. Beta-galactosidase staining was performed using the β-GalStaining Kit (Invitrogen Corporation, Carlsbad, Calif.) according to themanufacturer's instructions.

[0701] The present invention provides a production and expression kitthat allows easy construction, production and use of nucleic acidmolecules comprising all or a portion of a lentiviral genome (e.g.,lentiviral vectors). Aspects of the invention include, but are notlimited to, 1) directional TOPO® and GATEWAY™ Destination pLenti6/V5vectors with a useful selectable marker and epitope tag, 2) optimizedvirus production conditions and cell lines to reproducibly achieve >10⁵infectious viral particles per ml, 3) stable gene delivery andexpression of at least two genes into actively dividing mammalian cells,and 4) transduction of at least two non-dividing cell types.

[0702] A four plasmid co-transfection is used to create infectiouslentiviral vectors (Dull, et al., (1998) J. Virol. 72:8463-8471). One ofthe vectors (pLenti6/V5-DEST, pLenti6/V5-D-TOPO®, pLenti4/V5-DEST, orpLenti6/UbC/V5-DEST) contains the gene of interest and is packaged intothe virions (for vector maps, see FIGS. 36A-D). The other three plasmidsare co-transfected to supply the viral proteins in trans. None of thesethree vectors are packaged into the virions. Each vector and adescription of its features is described in more detail below. Vectormaps are provided as FIGS. 37A, 37B, and 37C.

[0703] pLenti6/V5-DEST or pLenti6/V5-D-TOPO carries gene of interest andblasticidin resistance gene and is packaged into viral particles. Thevector contains the RSV promoter, which enhances production of the viralgenomic RNA in the producer cell and removes dependence on HIV tatprotein. The vector also contains viral 5′ and 3′ LTRs (Long TerminalRepeats), which are required for viral packaging and reversetranscription of the viral RNA. The 3′ LTR also contains polyA signal.The vector contains the Ψ (psi) packaging signal. Nuclear export ofunspliced viral genomic RNA in the presence of rev occurs as a result ofthe RRE (Rev-Responsive Element) present in the vector. The vector alsoincorporates 5′ and 3′ splice sites that result in the removal of psiand RRE making expression of the gene of interest no longerrev-dependent in the host cell. The vector also contains Delta U3, a 400bp deletion in the 3′ LTR that gets copied to the 5′ LTR after reversetranscription of the viral genome in the transduced target cell. Thisresults in “self-inactivation” of the 5′ LTR for biosafety.

[0704] pLP1 expresses HIV-1 gag and pol genes in trans and is notpackaged into viral particles produced with this system. The plasmidcontains the RRE, which makes expression of gag/pol genes rev-dependent(for safety purposes).

[0705] pLP2 expresses HIV-1 rev gene in trans and, like pLP1, is notpackaged into viral particles. The plasmid encodes the rev protein,which is required for gag/pol expression and for nuclear export of theunspliced viral genome (from pLenti6/v5-DEST or D-TOPO®) for packaginginto the virions.

[0706] pLP/VSVG expresses the VSV-G envelope gene in trans. The plasmidis not packaged into viral particles, however, the VSV-G protein isincorporated into the viral particle. VSV-G is a non-HIV envelope thatbroadens the host range and stabilizes the viral particles (Yee 1994).

Results and Discussion

[0707] Vector construction. The vector pRRLsin.hCMV.GFPpre was used asthe starting material from Cell Genesys. This vector contains theessential elements for lentiviral packaging (e.g., 5′ and 3′ LTRs, psipackaging signal, rev responsive element (RRE) and necessary splicesites; see above for descriptions). In addition, it contains a deletionin the 3′ LTR (called “delta U3”) that results in a self-inactivation ofthe 5′ LTR after integration of the viral genome into the genome of thetarget cell (Dull 1998, Zufferey et al., (1998) J. Virol. 72:9873-80).This is an additional safety measure (see “Safety” section below) andhas no effect on vector performance since the 5′ LTR is only neededduring viral production, not gene expression in the target cell(Zufferey 1998). Finally, all polyadenylation (polyA) functions aresupplied by the 3′ LTR. The 3′ LTR serves as the polyA for the viralgenome (driven by the RSV/5′ LTR), the CMV promoter (gene of interest)and the SV40 promoter (blasticidin resistance). No heterologous polyAsignals should ever be included between the LTRs or viral productionwill be severely compromised due to transcription termination prior tothe 3′ LTR. The downstream SV40 polyA in the pLenti6/V5 vectors simplyenhances viral genomic RNA production in the producer cells and is notpackaged into the virions.

[0708] TOPO adaptation and purification. Fifty micrograms ofTOPO-charged pLenti6/V5-D-TOPO® was loaded on the Q-column and fractionscontaining the purified vector were collected in seven 0.5 ml fractions.The peak fraction (fraction 41) contained ˜20 μg of DNA by Hoechst (H33258) dye DNA fluorescence characterization and was eluted of at ˜500mM NaCl. Only this fraction was analyzed, however fractions 39-45 alsocontained TOPO-charged DNA. The fractions were diluted in 2× TOPOdilution buffer, so fraction 41 contained vector at ˜20 ng/μl finalconcentration. TOPO transformation results, using fraction 41 in twoexperiments (one with 750 bp insert, one with lacZ-alpha), are shown inTable 24. TABLE 24 pLenti6/V5-D-TOPO ® transformations. #colonies/Vector Insert μl vector Orientation (% correct) % background pLenti6/None 162 612 — — — — V5-D-TOPO 750 bp test 1665 — 9/10 (90%) — 9.7% —LacZ alpha — 4464 — 17/18 (94%) — 13.7%

[0709] Vector instability. While performing manipulations on thevectors, it was discovered that the presence of 182 basepairs of directrepeat present in the LTRs was triggering homologous recombination whentransformed into TOP10 and plated on LB-amp. This resulted in a visiblecolony phenotype. In clones where LTR recombination occurred thecolonies were large, while unrecombined (correct) clones resulted insmall colonies. FIG. 38 shows the results of an experiment in which twoLR reactions were performed with either pLenti6/V5-DEST alone orpLenti/V5-DEST plus pENTR/CAT and 3 μl of each was transformed intoTOP10 cells. 100 μl of the transformations were plated on regular LB-ampplates (No Bsd in FIG. 38) or LB-amp containing 50 μg/ml blasticidin.After overnight incubation at 37 degrees, colonies were photographed(FIG. 38A) and counted (FIG. 38B). Twenty-four clones (twelve each fromtwo independent experiments) from the DEST+CAT plates (+/−Bsd) wererandomly picked and screened by restriction digest to determine thepercentage of correct clones.

[0710] Since blasticidin resistance (driven by the EM7 promoter) ispresent between the LTRs, it was found that spreading blasticidin on thebacterial plate (to a final concentration of 50 μg/ml) resulted in allsmall colonies, none of which contained the LTR recombination product(not shown). This was further confirmed when GATEWAY™ LR reactions wereperformed using pLenti6/V5-DEST with and without including the pENTR-CATplasmid (FIG. 38B). Without blasticidin in the plate, backgroundcolonies arose from the DEST vector alone and only 50% of the DEST+CATclones were intact. However, when blasticidin was included in the plate,the DEST vector alone gave no background colonies and all DEST+CATclones were correct and intact (FIG. 38B). Therefore, it is recommendedthat one of two approaches be use when introducing a gene of interestinto pLenti6/V5 vectors: 1) if high efficiency cloning (i.e.library-scale) is not required, simply pick only the small colonies forminiprep analysis; or 2) to ensure ˜100% correct clones, includeblasticidin (50 μg/ml) in the bacterial plate following transformation.It has been observed that once a clone is isolated and shown to beintact, it appears to remain stable over multiple rounds of large-scalepropagation without blasticidin. Nevertheless, it is recommended thateach DNA preparation be verified by restriction digest prior toproceeding to virus production.

[0711] Transient transfection expression testing. To verify proteinexpression and the functionality of the V5 epitope tag, the lacZ ORF(with or without a stop codon) was GATEWAY™ cloned into pLenti6/V5-DEST.The resulting attB expression clones were transiently transfected intoCOS cells and analyzed by anti-β-galactosidase and anti-V5 westernblotting (FIG. 39). COS-7 cells were transiently transfected with:

[0712] Lane 1: mock; Lane 2: pcDNA3.1/V5His/lacZ; Lane 3:pLenti6/V5-GW-lacZ (no stop); Lane 4: pLenti6/V5-GW-lacZ (with stop);and lysates were analyzed by anti-lacZ or anti-V5 western blotting asindicated.

[0713] Compared to pcDNA3.1/V5His/lacZ, pLenti6/V5-GW/lacZ expressedequally well with and without the V5 epitope tag. In addition, lacZ (nostop) resulted in an efficiently expressed V5-tagged fusion protein(lane 3). This vector can be used as an expression control vector andmay be included in kits of the invention.

[0714] Virus production optimization. Previous reports had indicatedthat virus production is maximal in human 293 cells that express theSV40 large T antigen (Naldini, et al., (1996) Proc. Natl. Acad. Sci. USA93:11382-11388). Virus production was tested in severalneomycin-resistant 293FT clones.

[0715] These cell lines were created by stably transfecting 293F cellswith the pCMV/Sport6-T antigen plasmid in which the SV40 origin had beendeleted. 293FT clone #42 was found to produce the highest levels ofinfectious virus. The expression of the SV40 large T antigen wasconfirmed by western blot analysis and producer cell stocks werepropagated in G418 to maintain the large T antigen expression.

[0716] Since the production of virus requires a quadruple transfection,the importance of the ratio of the four plasmids was tested. Publishedreports suggested a variety of ratios as “optimal” (Dull 1998; Naldini1996; Mochizuki et al., (1998) J. Virol. 72:8873-8883), so eachpublished ratio was evaluated and compared to the simple 1:1:1:1. Littledifference was seen between the simple 1:1:1:1 and the more elaborateratios (e.g. 4:2.6:1:1.4). The highest and most reproducible titers weregenerated using a simple ratio of 1:1:1:1. The most effective timecourse for production of virus was determined. Various genes were clonedinto pLenti6/V5 and virus was produced in 293FT cells according to thefollowing optimized protocol

[0717] Day 0 Plate 5×10⁶ 293FT per 100 mm plate

[0718] Day 1 Four plasmid co-transfection (ratio=1:1:1:1)

[0719] 12 μg DNA total (3 μg each)

[0720] 36 μl Lipofectamine 2000

[0721] Day 2 Replace media

[0722] Day 3-4 Harvest supernatant containing virus

[0723] Spin 3000 rpm×15′ and/or filter 0.45 μm

[0724] Aliquot supernatant, use for titering and store −80° C.

[0725] Independent virus productions, of either the empty vector(pLenti6/V5-DEST) or carrying lacZ, GFP, CAT or protein kinase C, weretitered on HT1080 cells by counting the number of resultingblasticidin-resistant colonies generated per ml of supernatant and theresults are shown in FIG. 40. The optimized protocol which included highdensity plating of the 293FT cells (5×10⁶ cells per 100 mm plate) andthe optimal lipid to DNA ratio using Lipofectamine 2000. It was foundthat viral supernatants can be harvested either 2 or 3 days posttransfection with minimal differences in viral yield. Presumably, theshort half-life of the virus in culture media at 37° C. negates anyadvantage of viral accumulation over one extra day. For storage,aliquotting viral stocks at −80° C. is recommended. Anywhere from 0 to10% loss of viral titer for each freeze/thaw cycle of crude supernatantwas observed.

[0726] The size of the inserted gene of interest can affect the viraltiter. Three different genes were GATEWAY™ cloned into pLenti6/V5-DEST(lacZ, CAT and protein kinase C) and one gene was directionally TOPOcloned (GFP). Viral production was compared between these fourgene-containing vectors and an empty vector, pLenti6/V5 (FIG. 40).Averages from three independent experiments showed that the empty vectoryielded the highest viral titer (average 1.4×10⁷ cfu/ml), while thelargest insert (lacZ) yielded the lowest titers (average 4.7×10⁵pfu/ml). Inserted genes of intermediate size (GFP, CAT and PKC) yieldedtiters somewhere in between (4×10⁶, 9×10⁶ and 3×10⁶; respectively).These data indicated that both the GATEWAY™ and TOPO versions of thesevectors can produce viral supernatants that easily exceed a viral titerof 10⁵, even with the large lacZ gene. The wild type HIV-1 genome isapproximately 10 kb and the elements present in pLenti6/V5 vectors addup to 3.7 kb. Therefore, the theoretical gene-packaging limit isapproximately 6 kb.

[0727] Viral gene delivery and expression. The ability of the lentiviralvectors to deliver and express a variety of genes was furtherinvestigated. HT1080 cells were transduced with either Lenti6/V5-GW/lacZvirus (GATEWAY™) or Lenti6/V5-dT/GFP virus (D-TOPO®) and selected for 10days with 10 μg/ml blasticidin. LacZ was visualized using the β-GalStaining kit and GFP was visualized using the fluorescent microscope(FIG. 41). Both the GATEWAY™ lacZ and the D-TOPO® GFP vectorsefficiently generated heterogeneous pools of stably transduced cells inwhich nearly 100% of the cells expressed the heterologous gene. Inaddition to HT1080, HeLa and CHO cells have been stably transduced withsimilar efficiencies and levels of gene expression.

[0728] To confirm the above results, and to verify that a functional V5epitope tag was efficiently added to the expressed proteins, celllysates were prepared from HT1080 cells stably transduced with eitherthe lacZ, CAT, GFP or protein kinase C viruses (FIG. 42). HT1080 cellswere transduced in duplicate with lentiviral vectors carrying genes foreither lacZ, CAT, GFP or PKC and selected for 10 days with 10 μg/mlblasticidin. Cell lysates were analyzed by anti-V5 western blotting.Molecular weight markers and each V5 fusion protein are indicated.*indicates background V5 band. All four proteins are efficientlyexpressed and all are properly fused to a detectable V5 epitope. Inaddition, the delivery and efficient expression of protein kinase C (a“relevant” gene, i.e., not lacZ, GFP or CAT) indicates the robustnessand broad applicability of this virus production system.

[0729] Gene expression is correlated to MOI. Theoretically, themultiplicity of infection (MOI=number of virus per cell) shouldcorrelate with gene delivery and expression. To investigate this, HT1080cells were transduced in duplicate at various MOIs, ranging from 0.05 to1 (FIG. 43). HT1080 cells were transduced in duplicate withLenti6/V5-GW/lacZ virus at multiplicities of infection (MOI) of 0.05,0.1, 0.5 and 1. Forty-eight hours later, cells were either stained forβ-Gal (FIG. 43A) or harvested and analyzed for β-Gal activity (FIG.43B). As the β-Gal staining indicates, an increasing number of cellsbecome lacZ-positive as the MOI increases. At an MOI of 1, greater than80% of the cells express lacZ. At higher MOIs (e.g. MOI 5), 100% of thecells were transduced. When cell lysates were analyzed for lacZactivity, a near-linear dose response was observed as the MOI increasedfrom 0.05 to 1 (FIG. 43B). At higher MOIs (e.g. MOI 5), the lacZactivity continues to increase, but graph tends to flatten out.

[0730] Lentiviral transduction of non-dividing cells. One of the keyadvantages of lentiviruses over traditional retroviruses is that theyare capable of stably transducing non-dividing cells. This significantlyexpands the potential tranducible target cells to include: 1) growth- ordrug-arrested cells in culture, 2) non-dividing primary cell cultures,and 3) animals/tissues. To verify that lentiviral vectors of theinvention could perform under these conditions, they were using threedifferent approaches.

[0731] Drug-arrested cells. Actively-growing cells in culture can bearrested at specific phases of the cell cycle using a variety of drugs.This approach is widely used in cell cycle analysis and tumor biology.One commonly used drug, aphidicolin, reversibly binds to DNA polymerasedelta and is used to arrest cells at the G1/S transition (Seki et al.,(1980) Biochem Biophys Acta 610:413). To test the activity of lentiviralvectors of the invention under conditions of cell cycle arrest,aphidicolin-blocked HT1080 cells were transduced with Lenti6/V5-GW/lacZvirus (FIG. 44A). HT1080 cells were either actively growing or growtharrested at G1/S by aphidicolin and transduced at an MOI of 1, induplicate, with either rKAT6/V6-GW/lacZ retrovirus or Lenti6/V5-GW/lacZlentivirus. Forty-eight hours post transduction, cell lysates wereanalyzed for beta-galactosidase activity. The control virus,rKAT6/V5-GW/lacZ virus, is a traditional Moloney-based retroviruscarrying the same lacZ gene. Both retrovirus and lentivirus were capableof transducing actively growing cells, but only the lentiviral vectorwas capable of transducing the non-dividing culture.

[0732] Quiescent primary cells. The second approach was to apply thelentiviral vectors to non-dividing primary human cultures. A low-passageprimary human foreskin fibroblast culture (MJ90, Grand Island) wasplated into 6-well format and allowed to grow to confluence. Primaryfibroblasts are strongly contact inhibited and can be maintained formany weeks arrested in quiescence (G₀) when maintained as a confluentculture. Contact-inhibited non-dividing quiescent primary human foreskinfibroblasts were transduced with retrovirus (rKAT6/V5-GW/lacZ) andlentivirus (Lenti6/V5-GW/lacZ) at an MOI of 1 and β-Gal stainedforty-eight hours post transduction. Similar to the results inaphidicolin-arrested cells, only the lentiviral vector (and not theretroviral rKAT vector) was capable of transducing non-dividing cells.Approximately 50% of the quiescent primary cells were transduced with anMOI of 1 (FIG. 44B).

[0733] Post-mitotic primary neurons. Neuronal research is one area wherelentiviral vectors can offer significant advantages over other genetransfer methods. Neuronal cultures are typically non-dividing,“post-mitotic” cells that transfect poorly. Traditional Moloneyretroviruses are not useful since the cells never go through mitosis.Lentiviral vectors are one solution to overcome these hurdles, andvectors of the invention were tested to determine if they could stablytransduce these cells. Primary, post-mitotic rat neuronal tissues(cortical and hippocampal) were received from BrainBits, Inc. and thenprocessed and plated. Four days after plating, cells were transduced atan MOI of 1 with either Lenti6/V5-GW/lacZ lentivirus or rKAT6/V5-GW/lacZretrovirus. Three days post-transduction, cultures were stained forβ-galactosidase. All wells transduced with the lentiviral vectorsstained blue, with approximately 50% of the cells expressing detectableβ-galactosidase. Conversely, wells transduced with the rKAT retrovirusdid not show any β-galactosidase expression. These results indicatedthat lentiviruses of the invention effectively transduced post-mitoticneurons of either cortical or hippocampal origin.

[0734] Long-term gene expression from lentiviral vectors. The stabilityof gene expression after delivery by lentiviral transduction was tested.HT1080 cells were transduced with either the Lenti6/V5-GW/lacZlentivirus or the rKAT6/V5-GW/lacZ retrovirus and stably selected with10 μg/ml blasticidin. Cultures were maintained in blasticidin and wereβ-Gal stained at 10 days (FIG. 45A) and 6 weeks (FIG. 45B) posttransduction. No loss of gene expression was observed over 6 weeks inculture, indicating that lentiviral gene delivery is stable and geneexpression is persistent even at 6 weeks post transduction.

[0735] The present invention describes the generation of infectiouslentiviral particles based on the genome and lifecycle of HIV-1.Considerable effort has been put into developing a system that is safeto use and is as far-removed from wild type HIV as possible. Key safetyfeatures built into this “3^(rd) generation” system are as follows:

[0736] The viral particles produced in this system are replicationincompetent and only carry the gene(s) of interest. No other viralspecies are produced. This also means that none of the structural HIVgenes (necessary for production of viral progeny) are present in thepackaged viral genome. Only sequences flanked by the viral LTRs will bepackaged into virions (i.e., pLenti6/V5 vector). None of the threepackaging plasmids contain LTRs (FIGS. 37A-C); so while they areexpressed in the producer cell, they are never packaged into thevirions. Once a cell is infected (the proper term for this event is“transduced”), the only genes that are delivered and expressed are thegene of interest and the selectable marker. Gag, pol, rev and envelopegenes are not present in the viral genome and are therefore neverexpressed in the target cell, so no new virus can be produced.

[0737] The system described above is a four-plasmid system. Thenecessary HIV-1 genes (gag-pol and rev) have been separated ontoindividual plasmids, and the non-HIV envelope is on a third plasmid(FIGS. 37A-C). All four plasmids have been engineered not to contain anyregions of homology with each other to prevent unwanted recombinationevents that could lead to the generation of a replication competentvirus (Dull 1998). In other words, multiple non-homologous recombinationevents would need to occur to get all necessary components into oneviral genome. In addition, the expression of gag and pol (from pLP1) isrev-dependent, by virtue of the RRE in the gag/pol transcript. Thisprevents unwanted gag/pol expression if rev is not present (Dull 1998).In other embodiments, one or more of the genes necessary for generationof a replication-incompetent retrovirus according to the methods of theinvention (i.e., gag, pol, rev, and a pseudotyping envelope protein) maybe expressed from the genome of a host cell. Thus, some or all of thenecessary genes may be expressed from plasmids and some or all of thenecessary genes may be expressed from the host cell genome. In aparticular embodiment, one or more of the necessary genes may beexpressed from the host cell genome and at least one gene expressed fromthe host cell genome may be operably linked to an inducible promoter. Inanother embodiment, all the genes necessary may be expressed from thegenome of a host cell and one or more may be operably linked to aninducible promoter. When more than one gene is operably linked to aninducible promoter, the inducible promoters may be the same ordifferent.

[0738] The gene transfer vector pLenti6/V5 has been modified to be“self-inactivating” (Yu et al., (1986) Proc. Natl. Acad. Sci. USA83:3194-3198, Yee et al., (1987) Proc. Natl. Acad. Sci. USA84:5197-5201, Zufferey 1998). A deletion has been made in the 3′ LTR(called “delta U3”) that has no effect on the generation of viral genomefor packaging in the producer cell. However once the produced virustransduces a target cell, the mechanisms of reverse transcription usethe 3′ LTR as a template to create the 5′ LTR. The end result is anintegrated viral genome that is defective in both its 5′ and 3′ LTRs,and is no longer capable of producing packagable viral genome. Thismeans that transduction with lentiviral vectors of the invention doesnot generate a productive infection, instead ending with a gene ofinterest integrated into the host cell genome.

[0739] Despite all of these safety features, the lentivirus producedwith this system can still pose a biohazardous risk. As shown above,they are fully capable of transducing primary human cells, thus theseviruses should be treated as Biosafety Level 2 organisms. Extra careshould be taken when creating viruses carrying harmful or toxic genes(such as activated oncogenes). For further information on BL-2guidelines and lentivirus handling, please refer to: “Biosafety inMicrobiological and Biomedical Laboratories”, 4^(th) Ed. Centers forDisease Control and contact the CDC.

[0740] Conclusions. The lentivirus production and expression system ofthe invention is based on the 3^(rd) Generation lentiviral systemcreated at Cell Genesys (Dull 1998). This system allows one skilled inthe art to rapidly clone their gene of interest into a packagablelentiviral vector, via GATEWAY™ or directional TOPO, and providesmaterials necessary for the creation of infectious viral particles.Finally, these viruses are capable of stably delivering a variety ofgenes to both actively dividing and non-dividing primary andimmortalized human cell lines.

Example 10

[0741] Materials and methods of the present invention (e.g., theViraPower™ Lentiviral Expression System) allow creation of areplication-incompetent retroviruses (e.g., an HIV-1-based lentivirus),which can then be used to deliver and express a sequence of interest ineither dividing or non-dividing eukaryotic (e.g., mammalian) cells. Insome embodiments, materials of the present invention may include, butare not limited to, expression plasmids, for example, an expressionplasmid that contains the sequence of interest under the control of asuitable promoter (e.g., the human cytomegalovirus (CMV) immediate-earlyenhancer/promoter; see Andersson, et al. (1989) J. Biol. Chem. 264,8222-8229; Boshart, et al. (1985) Cell 41, 521-530; Nelson, et al.(1987) Molec. Cell. Biol. 7, 4125-4129) and also contains elements thatallow packaging of the construct into virions. Other materials suitablefor the practice of the present invention include an optimized mix ofpackaging plasmids (e.g., pLP1, pLP2, and pLP/VSVG) which may supply thestructural and replication proteins in trans that are required toproduce a recombinant retrovirus. In some embodiments, the presentinvention provides a cell line (e.g., 293FT), which allows production ofthe lentivirus following cotransfection of the expression plasmid andthe plasmids in the packaging mix. In some embodiments, the presentinvention provides a control expression plasmid containing the lacZ genewhich, when packaged into virions and transduced into a mammalian cellline, expresses β-galactosidase.

[0742] Using the materials and methods of the present invention (e.g.,the ViraPower™ Lentiviral Expression System) to facilitateretroviral-based expression of the gene of interest provides thefollowing advantages: 1) generates an HIV-1-based lentivirus thateffectively transduces both dividing and non-dividing mammalian cells,thus broadening the potential applications beyond those of traditionalMoloney Leukemia Virus (MoMLV)-based retroviral systems (Naldini, 1998);2) efficiently delivers the gene of interest to mammalian cells inculture or in vivo (Dull et al., 1998); 3) provides stable, long-termexpression of a target gene beyond that offered by traditionaladenoviral-based systems (Dull et al., 1998; Naldini et al., 1996); 4)produces a pseudotyped virus with a broadened host range (Yee et al.,1994); and 5) includes multiple features designed to enhance thebiosafety of the system.

[0743] One of skill in the art can use the teachings provided herein to:co-transfect the vectors described herein (e.g., pLenti6/V5-basedexpression vector) and the ViraPower™ Packaging Mix into the 293FT cellline to produce a lentiviral stock; titer the lentiviral stock; use thelentiviral stock to transduce a mammalian cell line of choice; assay for“transient” expression of one or more recombinant proteins encoded bythe transduced vector; and/or generate a stably transduced cell line, ifdesired.

[0744] Additional details and instructions to generate an expressionvector using pLenti6/V5-D-TOPO® or pLenti6/V5-DEST™ are available (e.g.,pLenti6/V5 Directional TOPO® Cloning Kit manual, catalog no. K4955-10,version B, or pLenti6/V5-DEST™ GATEWAY™ Vector Pack manual, catalog nos.V496-10, V498-10, and V499-10, version C, Invitrogen Corporation,Carlsbad, Calif.). For instructions to culture and maintain the 293FTproducer cell line, see Example 13 below.

[0745] Expression systems of the present invention (e.g., the ViraPower™Lentiviral Expression System) facilitate highly efficient, in vitro orin vivo delivery of a target gene to dividing and non-dividing mammaliancells using a replication-incompetent lentivirus. Based on the lentikat™system developed by Cell Genesys (Dull et al., 1998), the ViraPower™Lentiviral Expression System possesses features which enhance itsbiosafety while allowing high-level gene expression in a wider range ofcell types than traditional retroviral systems.

[0746] One component of the systems of the invention is an expressionvector (e.g., a pLenti6/V5-based expression vector) into which thesequence of interest (e.g., encoding a gene of interest) will be cloned.Expression of the sequence of interest is controlled by a promoter ofchoice, for example, the human cytomegalovirus (CMV) promoter. Thevector also contains the elements required to allow packaging of theexpression construct into virions (e.g. 5′ and 3′ LTRs, Ψ packagingsignal).

[0747] Another component of a system of the invention is one or moreplasmids encoding the activities necessary for packaging the RNAproduced from the expression vector (e.g., the ViraPower™ Packaging Mixthat contains an optimized mixture of the three packaging plasmids,pLP1, pLP2, and pLP/VSVG). These plasmids supply the helper functions aswell as structural and replication proteins in trans required to producethe lentivirus.

[0748] An optional component of the system is an optimized cell line(e.g., the 293FT producer cell line) that may stably express the SV40large T antigen.

[0749] Expression of the SV40 large T antigen may be under the controlof any promoter known in the art, for example, the human CMV promoter.Expression of the large T antigen facilitates optimal production ofvirus.

[0750] In an embodiment, plasmids containing the packaging activities(e.g., the ViraPower™ Packaging Mix) and an expression plasmid (e.g.,the pLenti6/V5 vector containing a sequence of interest) may beco-transfected into a suitable host cell line (e.g., 293FT cells) toproduce a replication-incompetent lentivirus, which can then betransduced into the mammalian cell line of interest. Once the lentivirusenters the target cell, the viral RNA is reverse-transcribed, activelyimported into the nucleus (Lewis et al. 1994; Naldini et al., 1999), andstably integrated into the host genome (Buchschacher et al., 2000;Luciw, (1996) In Fields Virology, B. N. Fields, et al. eds.(Philadelphia, Pa.: Lippincott-Raven Publishers), pp. 1881-1975). Oncethe lentiviral construct has integrated into the genome, transientexpression of a recombinant protein can be assayed or blasticidinselection can be used to generate a stable cell line for long-termexpression.

[0751] Most retroviral vectors are limited in their usefulness as genedelivery vehicles by their restricted tropism and generally low titers.In the systems of the invention (e.g., the ViraPower™ LentiviralExpression System), this limitation has been overcome by use of the Gglycoprotein gene from Vesicular Stomatitis Virus (VSV-G) as apseudotyping envelope, thus allowing production of a high titerlentiviral vector with a significantly broadened host cell range (Bumset al., (1993) Proc. Natl. Acad. Sci. USA 90, 8033-8037, Emi et al.,(1991) J. Virol. 65, 1202-1207, Yee et al., 1994). Cell Lines and CellTypes Tested Cell Line or Cell Type Description Condition Tested 293Human embryonic kidney Actively dividing (Graham et al., (1977) J. Gen.Virol. 36, 59-74) HT1080 Human fibrosarcoma Actively dividing (Rasheedet al., Aphidicolin-arrested (1974) Cancer 33, (at the G1/S transi-1027-1033) tion) HeLa Human cervical adeno- Actively dividing carcinomaCHO-K1 Chinese hamster ovary Actively dividing (Kao et al., (1968) Proc.Natl. Acad. Sci. USA 60, 1275-1281) Primary foreskin Human foreskinContact inhibited, fibroblasts growth-arrested (in G₀) Primary hippo-Rat neuronal tissue Non-dividing, post- campal neurons mitotic Primarycortical Rat neuronal tissue Non-dividing, post- neurons mitotic

[0752] The present invention is suitable for in vivo gene deliveryapplications. Many groups have successfully used lentiviral vectors toexpress a target gene in tissues including brain, retina, pancreas,muscle, liver, and skin (Gallichan et al., (1998) Human Gene Therapy 9,2717-2726; Kafri et al., (1997) Nature Genetics 17, 314-317; Miyoshi etal., (1997) Proc. Natl. Acad. Sci. USA 94, 10319-10323; Naldini, (1998)Curr. Opin. Biotechnol. 9, 457-463; Pfeifer et al., (2001) Proc. Natl.Acad. Sci. USA 98, 11450-11455; Pfeifer et al., (2001) Mol. Ther. 3,319-322; Takahashi et al., (1999) J. Virol. 73, 7812-7816). For moreinformation about target genes that have been successfully expressed invivo using lentiviral-based vectors, refer to the references above aswell as the following additional references (Baek et al., 2001; Dull etal., 1998; Park et al., 2001; Peng et al., 2001).

[0753] The systems of the invention (e.g., the ViraPower™ LentiviralExpression System) are third-generation systems based on lentiviralvectors developed by Dull et al. (1998). These third-generationlentiviral systems include a significant number of safety featuresdesigned to enhance their biosafety and to minimize their relation tothe wild-type, human HIV-1 virus. These safety features are discussedbelow.

[0754] The expression vector (pLenti6/V5-D-TOPO® or pLenti6/V5-DEST™)contains a deletion in the 3′ LTR (ΔU3) that does not affect generationof the viral genome in the producer cell line, but results in“self-inactivation” of the lentivirus after transduction of the targetcell (Yee et al., 1987; Yu et al., 1986; Zufferey et al., 1998). Onceintegrated into the transduced target cell, the lentiviral genome is nolonger capable of producing packageable viral genome.

[0755] The number of genes from HIV-1 that are used in the system hasbeen reduced to three (i.e. gag, pol, and rev).

[0756] The VSV-G gene from Vesicular Stomatitis Virus is used in placeof the HIV-1 envelope (Bums et al., 1993; Emi et al., 1991; Yee et al.,1994).

[0757] Genes encoding the structural and other components required forpackaging the viral genome are separated onto four plasmids. All fourplasmids have been engineered not to contain any regions of homologywith each other to prevent undesirable recombination events that couldlead to the generation of a replication-competent virus (Dull et al.,1998).

[0758] Although the three packaging plasmids allow expression in transof proteins required to produce viral progeny (e.g. gal, pol, rev, env)in the 293FT producer cell line, none of them contain LTRs or the Ψpackaging sequence. This means that none of the HIV-1 structural genesare actually present in the packaged viral genome, and thus, are neverexpressed in the transduced target cell. No new replication-competentvirus can be produced.

[0759] The lentiviral particles produced in this system arereplication-incompetent and only carry the gene of interest. No otherviral species are produced.

[0760] Expression of the gag and pol genes from pLP1 has been renderedRev-dependent by virtue of the HIV-1 RRE in the gag/pol mRNA transcript.Addition of the RRE prevents gag and pol expression in the absence ofRev (Dull et al., 1998).

[0761] A constitutive promoter (RSV promoter, Gorman et al. (1982).Proc. Natl. Acad. Sci. USA 79, 6777-6781) has been placed upstream ofthe 5′ LTR in the pLenti6/V5 expression vector to offset the requirementfor Tat in the efficient production of viral RNA (Dull et al., 1998).

[0762] Despite the inclusion of the safety features discussed above, thelentivirus produced with the systems of the invention can still posesome biohazardous risk since they can transduce primary human cells. Forthis reason, published guidelines for BL-2 should be followed.Furthermore, exercise extra caution when creating lentivirus carryingpotential harmful or toxic genes (e.g. activated oncogenes).

[0763] For more information about the BL-2 guidelines and lentivirushandling, refer to the document, “Biosafety in Microbiological andBiomedical Laboratories”, 4^(th) Edition, published by the Centers forDisease Control (CDC).

[0764] The diagram in FIG. 35 describes the general steps required toexpress a sequence of interest using an exemplary system of theinvention.

[0765] The present of the invention is designed to help one skilled inthe art create a lentivirus to deliver and express a gene of interest inmammalian cells. For more information about retroviral biology andeukaryotic cell culture, refer to the following published reviews:Buchschacher et al. (2000); Luciw (1996); Naldini (1999), Naldini(1998), and Yee (1999) Retroviral Vectors. In The Development of HumanGene Therapy, T. Friedmann, ed. (Cold Spring Harbor, N.Y.: Cold SpringHarbor Laboratory Press), pp. 21-45.

[0766] The pLP1, pLP2, pLP/VSVG plasmids are provided in an optimizedmixture to facilitate viral packaging of an expression vector (e.g., apLenti6/V5-based expression vector) following cotransfection into 293FTproducer cells. The amount of the packaging mix (195 μg) andLipofectamine™ 2000 transfection reagent (0.75 ml) supplied in the kitis sufficient to perform 20 cotransfections in 10 cm plates using therecommended protocol describe herein. To use the ViraPower™ PackagingMix, resuspend in 195 μl of sterile water to obtain a 1 μg/μl stock.

[0767] A pLenti6/V5 expression vector containing a gene of interest inpLenti6/V5-D-TOPO® or pLenti6/V5-DEST™ can be generated using methodsdescribed herein. Once an expression construct has been created, use anymethod of choice to prepare purified plasmid DNA. Plasmid DNA fortransfection into eukaryotic cells must be clean and free from phenoland sodium chloride as contaminants may kill the cells, and salt willinterfere with lipid complexing, decreasing transfection efficiency.Suitable methods for isolating plasmid of sufficient purity include theS.N.A.P.™ MidiPrep Kit (Invitrogen Corporation, Carlsbad, Calif.,Catalog no. K1910-01) and CsCl gradient centrifugation.

[0768] Resuspend the purified expression plasmid (e.g., a pLenti6/V5expression plasmid) containing a gene of interest in sterile water orTE, pH 8.0 at a concentration ranging from 0.1-3.0 μg/μl. 3 μg ofexpression plasmid may be used for each transfection.

[0769] A suitable host cell line is the human 293FT cell line availablefrom Invitrogen Corporation, Carlsbad, Calif. and supplied with theViraPower™ Lentiviral Expression kits (Naldini et al., 1996). The 293FTcell line, a derivative of the 293F cell line, stably and constitutivelyexpresses the SV40 large T antigen from pCMVSPORT6TAg.neo and must bemaintained in medium containing Geneticin®.

[0770] Before a stably transduced cell line expressing a gene ofinterest can be created, a lentiviral stock (containing the packagedexpression construct) must be created by cotransfecting the optimizedpackaging plasmid mix and an expression vector (e.g., a pLenti6/V5-basedexpression vector) into a suitable host cell line (e.g., the 293FT cellline).

[0771] One suitable protocol for generating a lentiviral stock employsthe following materials: ViraPower™ Packaging Mix (supplied with thekit; resuspend in 195 μl of sterile water to a concentration of 1μg/μl); pLenti6/V5 expression vector containing a gene of interest(0.1-3.0 μg/μl in sterile water or TE, pH 8.0); pLenti6/V5-basedpositive control vector (supplied with the kit; resuspend in sterilewater to a concentration of 1 μg/μl); 293FT cells cultured in theappropriate medium (see Example 13); Lipofectamine™ 2000 transfectionreagent (supplied with the kit; store at +4° C. until use); Opti-MEMO® IReduced Serum Medium (pre-warmed; see below); Fetal bovine serum (FBS);sterile 10 cm tissue culture plates (one each for the lentiviralconstruct, positive control, and negative control); sterile tissueculture supplies; 15 ml sterile, capped, conical tubes; and cryovials.

[0772] Each pLenti6/V5-based expression vector kit includes a positivecontrol vector for use as an expression control (e.g.pLenti6/V5-GW/lacZ). It is recommended that the positive control vectorbe included in a cotransfection experiment to generate a controllentiviral stock that may be used to help optimize expression conditionsin a mammalian cell line of interest.

[0773] Any suitable transfection reagent may be used to introduce theplasmids into the producer cell line. One suitable transfection reagentis Lipofectamine™ 2000 reagent (Ciccarone et al., (1999) Focus 21,54-55). This reagent is a proprietary, cationic lipid-based formulationsuitable for the transfection of nucleic acids into eukaryotic cells.Using Lipofectamine™ 2000 to transfect 293FT cells offers the followingadvantages: provides the highest transfection efficiency in 293FT cells;DNA-Lipofectamine™ 2000 complexes can be added directly to cells inculture medium in the presence of serum; and removal of complexes ormedium change or addition following transfection are not required,although complexes can be removed after 4-6 hours without loss ofactivity.

[0774] To facilitate optimal formation of DNA-Lipofectamine™ 2000complexes, a reduced serum medium (e.g., Opti-MEM® I Reduced SerumMedium available from Invitrogen Corporation, Carlsbad, Calif.) may beused.

[0775] Lentiviral stocks in 293FT cells produced using the optimizedtransfection conditions described herein. The amount of lentivirusproduced using these recommended conditions (at a titer of 1×10⁵ to1×10⁷ transducing units (TU)/ml) is generally sufficient to transduce1×10⁶ to 1×10⁸ cells at a multiplicity of infection (MOI)=1. ConditionAmount Tissue culture plate size 10 cm (one per lentiviral construct)Number of 293FT cells to 5 × 10⁶ cells (see below) transfect Amount ofViraPower ™ 9 μg Packaging Mix (9 μl of 1 μg/μl stock) Amount ofpLenti6/V5 3 μg expression plasmid Amount of Lipofectamine ™ 36 μl 2000

[0776] 293FT cells should be plated 24 hours prior to transfection incomplete medium, and should be 90-95% confluent on the day oftransfection. Make sure that cells are healthy at the time of plating.

[0777] Follow the procedure below to cotransfect 293FT cells. Rememberthat the cells may be kept in culture medium during transfection. Apositive control and a negative control (no DNA, no Lipofectamine™ 2000)are recommended to help evaluate results.

[0778] The day before transfection, trypsinize and count the 293FTcells, plating them at 5×10⁶ cells per 10 cm plate. Plate cells in 10 mlof normal growth medium containing serum.

[0779] On the day of transfection, remove the culture medium from the293FT cells and replace with 5 ml of normal growth medium containingserum (or Opti-MEM® I Medium containing serum). Do not includeantibiotics.

[0780] Prepare DNA-Lipofectamine™ 2000 complexes for each transfectionsample by performing the following: Dilute 9 μg of the optimizedpackaging mix and 3 μg of pLenti6/V5 expression plasmid DNA (12 μgtotal) in 1.5 ml of Opti-MEM® I Medium without serum. Mix gently. MixLipofectamine™ 2000 gently before use, then dilute 36 μl in 1.5 ml ofOpti-MEM® I Medium without serum. Mix gently and incubate for 5 minutesat room temperature. After the 5 minute incubation, combine the dilutedDNA with the diluted Lipofectamine™ 2000. Mix gently. Incubate for 20minutes at room temperature to allow the DNA-Lipofectamine™ 2000complexes to form. The solution may appear cloudy, but this will notimpede the transfection.

[0781] Add the DNA-Lipofectamine™ 2000 complexes dropwise to each plate.Mix gently by rocking the plate back and forth. Incubate the cellsovernight at 37° C. in a CO₂ incubator.

[0782] The next day, remove the medium containing the DNA-Lipofectamine™2000 complexes and replace with complete culture medium (i.e. D-MEMcontaining 10% FBS, 2 mM L-glutamine, and 1% penicillin/streptomycin).Expression of the VSV G glycoprotein causes 293FT cells to fuise,resulting in the appearance of multinucleated syncitia. Thismorphological change is normal and does not affect production of thelentivirus.

[0783] Harvest virus-containing supernatants 48-72 hoursposttransfection by removing medium to a 15 ml sterile, capped, conicaltube. Minimal differences in viral yield are observed whethersupernatants are collected 48 or 72 hours posttransfection. Rememberthat the supernatant contains infectious virus at this stage. Follow therecommended guidelines for working with BL-2 organisms.

[0784] Centrifuge at 3000 rpm for 15 minutes at +4° C. in a table topclinical centrifuge.

[0785] Perform A filtration step, if desired. Pipet viral supernatantsinto cryovials in 1 ml aliquots. Store viral. stocks at −80° C.

[0786] If the lentiviral construct is to be used for in vivoapplications or if the stock is to be concentrated to obtain a highertiter, filtering the viral supernatant through a sterile, 0.45 μm lowprotein binding filter after the low-speed centrifugation step isrecommended. A suitable filter is the Millex-HV 0.45 μm PVDF filter(Millipore, Catalog no. SLHVR25LS).

[0787] Place viral stocks at −80° C. for long-term storage. Repeatedfreezing and thawing is not recommended as it may result in loss ofviral titer. When stored properly, viral stocks of an appropriate titershould be suitable for use for up to one year. After long-term storage,it is recommended that the titer of the viral be determined beforetransducing a cell line of interest.

[0788] It is possible to scale up the cotransfection experiment toproduce a larger volume of lentivirus, if desired. For example, thecotransfection experiment may scaled up from a 10 cm plate to a T225flask and up to 50 ml of viral supernatant may be harvested. To scaleup, increase the number of cells plated and the amounts of DNA,Lipofectamine™ 2000, and medium used in proportion to the difference insurface area of the culture vessel.

[0789] Before proceeding to transduce the mammalian cell line ofinterest and express a recombinant protein, it is recommended that thetiter of the lentiviral stock be determined. While this procedure is notrequired for some applications, it is necessary to control the number ofintegrated copies of the lentivirus or to generate reproducibleexpression results.

[0790] To determine the titer of a lentiviral stock: prepare 10-foldserial dilutions of the lentiviral stock; transduce the differentdilutions of lentivirus into the mammalian cell line of choice in thepresence of Polybrene®; select for stably transduced cells usingblasticidin; and stain and count the number of blasticidin-resistantcolonies in each dilution.

[0791] A number of factors can influence viral titers. One factor is thesize of the sequence of interest inserted into the expression vector.Titers will generally decrease as the size of the insert increases. Thesize of the wild-type HIV-1 genome is approximately 10 kb. Since thesize of the elements required for expression from pLenti6/V5 totalsapproximately 4 kb, the size of the gene of interest shouldtheoretically not exceed 6 kb for efficient packaging.

[0792] Other factors that may influence viral titer are thecharacteristics of the cell line used for titering, the age of thelentiviral stock, the number of freeze thaw cycles that the stock hasundergone, and the storage conditions of the stock. Viral titers maydecrease with long-term storage at −80° C. If a lentiviral stock hasbeen stored for 6 months to 1 year, it is recommended that the titer bedetermined prior to use in an expression experiment. Viral titers candecrease as much as 10% with each freeze/thaw cycle. Lentiviral stocksshould be aliquotted and stored at −80° C.

[0793] The titer of a lentiviral stock may be determined using anymammalian cell line of choice. Generally, it is recommended that thesame mammalian cell line be used to titer the lentiviral stock will beused to perform expression studies. However, in some instances, adifferent cell line may be used to titer the lentivirus (e.g. ifperforming expression studies in a non-dividing cell line or a primarycell line). In these cases, suitable cell lines with which to titer thelentivirus are those that: grow as an adherent cell line; are easy tohandle; exhibit a doubling time in the range of 18-25 hours; and arenon-migratory. An example of a suitable cell is the HT1080 humanfibrosarcoma cell line (ATCC, Catalog no. CCL-121) for titeringpurposes, but other cell lines including HeLa and NIH3T3 are alsosuitable.

[0794] The titer of a lentiviral construct may vary depending on whichcell line is chosen. If more than one lentiviral construct are to beused, it is recommended that the titer all of the lentiviral constructsbe determined using the same cell line.

[0795] The pLenti6/V5 expression construct contains the blasticidinresistance gene (bsd) (Kimura et al., (1994) Biochim. Biophys. ACTA1219, 653-659, Izumi, et al. (1991) Exp. Cell Res. 197, 229-233.) toallow for blasticidin selection of mammalian cells that have stablytransduced the lentiviral construct (Takeuchi et al., (1958) The Journalof Antibiotics, Series A 11, 1-5; Yamaguchi et al., (1965) J. Biochem(Tokyo) 57, 667-677.

[0796] Since stably transduced cells are selected using blasticidin, theminimum concentration of blasticidin required to kill untransduced cellsmust be determined (i.e. perform a kill curve experiment). Typically,concentrations ranging from 2-10 μg/ml blasticidin are sufficient tokill most untransduced mammalian cell lines. For any given cell line ofinterest, a range of concentrations should be tested (see protocolbelow) to ensure that the minimum concentration necessary for the cellline is used. A suitable method to determine the appropriateconcentration of blasticidin for a given cell line follows.

[0797] Prepare a set of 6 plates. Plate cells at approximately 25%confluence. Allow cells to adhere overnight.

[0798] The next day, substitute culture medium with medium containingvarying concentrations of blasticidin (e.g., 0, 2, 4, 6, 8, 10 μg/mlblasticidin).

[0799] Replenish the selective media every 3-4 days, and observe thepercentage of surviving cells.

[0800] Determine the appropriate concentration of blasticidin that killsthe cells within 10 days after addition of blasticidin.

[0801] To determine the titer of a lentiviral construct, the followingmaterials will be needed: the lentiviral stock (store at −80° C. untiluse); an adherent mammalian cell line of choice; complete culture mediumfor the cell line; hexadimethrine bromide (Polybrene®; Sigma, Catalogno. H9268; 6-well tissue culture plates; blasticidin (10 mg/ml stocksolution); crystal violet (Sigma, Catalog no. C3886; prepare a 1%crystal violet solution in 10% ethanol); and Phosphate-Buffered Saline(PBS; Invitrogen, Catalog no. 10010-023).

[0802] When adding virus to mammalian cells, Polybrene® is included toenhance transduction of the virus into the cell. To use Polybrene®:prepare a 6 mg/ml stock solution in deionized, sterile water;filter-sterilize and dispense 1 ml aliquots into sterile microcentrifugetubes; store at −20° C. for long-term storage. Stock solutions may bestored at −20° C. for up to 1 year. Do not freeze/thaw the stocksolution more than 3 times as this may result in loss of activity. Theworking stock may be stored at +4° C. for up to 2 weeks.

[0803] The media contains infectious virus and appropriate safetyprecautions should be taken. For example, perform all manipulationswithin a certified biosafety cabinet. Treat media containing virus withbleach. Treat used pipets, pipette tips, and other tissue culturesupplies with bleach or dispose of as biohazardous waste. Wear gloves, alaboratory coat, and safety glasses or goggles when handling viralstocks and media containing virus.

[0804] Follow the procedure below to determine the titer of a lentiviralstock using the mammalian cell line of choice. At least one 6-well plateis used for every lentiviral stock to be titered (one mock well plusfive dilutions). If a lentiviral stock of the pLenti6/V5-GW/lacZpositive expression control has been made, it is recommended that thisstock be titered as well.

[0805] The day before transduction (Day 1), trypsinize and count thecells, plating them such that they will be 30-50% confluent at the timeof transduction. Incubate cells at 37° C. overnight.

[0806] Example: When using HT1080 cells, generally plate 2×10⁵ cells perwell in a 6-well plate.

[0807] On the day of transduction (Day 2), thaw the lentiviral stock andprepare 10-fold serial dilutions ranging from 10⁻² to 10⁻⁶. For eachdilution, dilute the lentiviral construct into complete culture mediumto a final volume of 1 ml. Do not vortex. A wider range of serialdilutions (10⁻² to 10⁻⁸) may be used, if desired.

[0808] Remove the culture medium from the cells. Mix each dilutiongently by inversion and add to one well of cells (total volume=1 ml).

[0809] Add Polybrene® to each well to a final concentration of 6 μg/ml.Swirl the plate gently to mix. Incubate at 37° C. overnight.

[0810] The following day (Day 3), remove the media containing virus andreplace with 2 ml of complete culture medium.

[0811] The following day (Day 4), remove the medium and replace withcomplete culture medium containing the appropriate amount of blasticidinto select for stably transduced cells.

[0812] Remove medium and replace with fresh medium containingblasticidin every 3-4 days.

[0813] After 10-12 days of selection (day 14-16), no live cells in themock well and discrete blasticidin-resistant colonies in one or more ofthe dilution wells should be seen. Remove the medium and wash the cellswith 2 ml of PBS. Repeat the wash.

[0814] Add 1 ml of crystal violet solution and incubate for 10 minutesat room temperature.

[0815] Remove the crystal violet stain and wash the cells with 2 ml ofPBS. Repeat wash.

[0816] Count the blue-stained colonies and determine the titer of thelentiviral stock.

[0817] When titering Lenti6/V5 lentiviral stocks using HT1080 cells,generally titers ranging from 5×10⁵ to 2×10⁷ transducing units (TU)/mlare observed. If the titer of a lentiviral stock is less than 1×10⁵TU/ml, a new lentiviral stock should be produced.

[0818] As an example, a Lenti6/V5-GW/lacZ lentiviral stock was generatedusing the protocols described herein. HT1080 cells were transduced with10-fold serial dilutions of the lentiviral supernatant (10⁻² to 10⁻⁶dilutions) or untransduced (mock) following the protocol describedabove. Forty-eight hours post-transduction, the cells were placed underblasticidin selection (10 μg/ml). After 10 days of selection, the cellswere stained with crystal violet (see plate below), and colonies werecounted. In the plate, the colony counts were: mock: no colonies; 10⁻²dilution: confluent; undeterminable, 10⁻³ dilution: confluent;undeterminable, 10⁻⁴ dilution: confluent; undeterminable, 10⁻⁵ dilution:46, and 10⁻⁶ dilution: 5. Thus, the titer of this lentiviral stock is4.8×10⁶ TU/ml (i.e. average of 46×10⁵ and 5×10⁶).

[0819] Once a lentiviral stock with a suitable titer has been generatd,the lentiviral construct may be transduced into the mammalian cell lineof choice and assayed for expression of a recombinant protein. An assayfor expression of a gene of interest may be conducted in the followingways:

[0820] 1) Pool a heterogeneous population of cells and test forexpression directly after transduction (i.e. “transient” expression).Note that 24-48 hours must elapse after transduction before harvestingcells to allow time for the lentivirus genome to reverse transcribe andintegrate into the chromosomal DNA. Integration must take place beforeexpression of the gene of interest can occur.

[0821] 2) Select for stably transduced cells using blasticidin. Thisrequires a minimum of 10-12 days after transduction, but allowsgeneration of clonal cell lines that stably express the gene ofinterest.

[0822] Stable expression of a target gene typically may be observed forat least 6 weeks following transduction and selection.

[0823] To select for stably transduced cells, the minimum concentrationof blasticidin required to kill the untransduced mammalian cell linemust be determined as described above. If the titer of the lentiviralconstruct was determined in the same cell line used to perform stableexpression experiment, then the same concentration of blasticidin may beused for selection as was used for titering.

[0824] To obtain optimal expression of a gene of interest, cells must betransduced with a suitable MOI of lentivirus. MOI is defined as thenumber of virus particles per cell and generally correlates with thenumber of integration events and as a result, expression. Typically,expression levels increase linearly as the MOI increases.

[0825] A number of factors can influence determination of an optimal MOIincluding the nature of the cell line (e.g. non-dividing vs. dividingcell type), its transduction efficiency, the application of interest,and the nature of the gene of interest. If transducing a lentiviralconstruct into a mammalian cell line of choice for the first time, arange of MOIs should be used (e.g. 0, 0.05, 0.1, 0.5, 1, 2, 5) todetermine the MOI required to obtain optimal expression of therecombinant protein for a particular application.

[0826] In general, 80-90% of the cells in an actively dividing cell line(e.g. HT1080, HeLa, CHO-K1) express a target gene when transduced at anMOI of ˜1. Some non-dividing cell types transduce lentiviral constructsless efficiently. For example, only about 50% of the cells in a cultureof primary human fibroblasts express a target gene when transduced at anMOI of ˜1. If transducing a lentiviral construct into a non-dividingcell type, it may be necessary to increase the MOI to achieve optimalexpression levels for a recombinant protein.

[0827] If a Lenti6/V5-GW/lacZ control lentiviral construct has beenconstructed, it may be used to help determine the optimal MOI for aparticular cell line and application. Once the Lenti6/V5-GW/lacZlentivirus has been transduced into the mammalian cell line of choice,the gene encoding β-galactosidase will be constitutively expressed andcan be easily assayed using standard techniques.

[0828] Remember that viral supernatants are generated by harvestingspent media containing virus from the 293FT producer cells. Spent medialacks nutrients and may contain some toxic waste products. If a largevolume of viral supernatant is used to transduce a mammalian cell line(e.g. 1 ml of viral supernatant per well in a 6-well plate), note thatgrowth characteristics or morphology of the cells may be affected duringtransduction. These effects are generally alleviated after transductionwhen the media is replaced with fresh, complete media.

[0829] It is possible to concentrate VSV-G pseudotyped retrovirusesusing a variety of methods without significantly affecting theirtransducibility. If the titer of a lentiviral stock is relatively low(less than 5×10⁵ TU/ml) and an experiment requires a large volume ofviral supernatant (e.g. a relatively high MOI), the virus may beconcentrated before proceeding to transduction. For details andguidelines to concentrate the virus, refer to published referencesources (Yee, 1999).

[0830] To transduce a selected cell line, the following materials willbe required: a titered stock of virus (e.g., a Lenti6/V5 lentiviralstock) which should be stored at −80° C. until use; a cell line ofchoice (e.g., a mammalian cell line); complete culture medium for thecell line; hexadimethrine bromide (Polybrene®; 6 mg/ml stock solution);appropriately sized tissue culture plates for the intended application;and blasticidin (if selecting for stably transduced cells; 10 mg/mlstock solution).

[0831] Follow the procedure below to transduce the mammalian cell lineof choice with a lentiviral construct.

[0832] Plate cells in complete media as appropriate for the intendedapplication.

[0833] On the day of transduction (Day 1), thaw the lentiviral stock anddilute (if necessary) the appropriate amount of virus (at a suitableMOI) into fresh complete medium. Do not vortex.

[0834] Remove the culture medium from the cells. Mix the mediumcontaining virus gently by pipetting and add to the cells.

[0835] Add Polybrene® to a final concentration of 6 μg/ml. Swirl theplate gently to mix. Incubate at 37° C. overnight. To reduce possiblenegative effects of transducing cells with undiluted viral stock, it ispossible to incubate cells for as little as 6 hours prior to changingmedium.

[0836] The following day (Day 2), remove the medium containing virus andreplace with fresh, complete culture medium.

[0837] The following day (Day 3), perform one of the following: harvestthe cells and assay for expression of the recombinant protein ofinterest if performing transient expression experiments; or remove themedium and replace with fresh, complete medium containing theappropriate amount of blasticidin to select for stably transduced cells.

[0838] Remove medium and replace with fresh medium containingblasticidin every 3-4 days until blasticidin-resistant colonies can beidentified (generally 10-12 days after selection).

[0839] Pick at least 5 blasticidin-resistant colonies and expand eachclone to assay for expression of the recombinant protein.

[0840] Note that integration of the lentivirus into the genome israndom. Depending upon the influence of the surrounding genomicsequences at the integration site, varying levels of recombinant proteinexpression may be seen from different blasticidin-resistant clones.Testing at least 5 blasticidin-resistant clones and selecting the clonethat provides the optimal expression of the recombinant protein ofinterest is recommended.

[0841] Any method of choice known to those skilled in the art may beused to detect a recombinant protein of interest including, but notlimited to, functional analysis, immunofluorescence, or western blot. Ifthe gene of interest is cloned in frame with an epitope tag, therecombinant protein may be detected in a western blot using an antibodyto the epitope tag.

[0842] Below are listed some potential problems and possible solutionsthat may help troubleshoot cotransfection and titering experiments.Problem Reason Solution Low viral Low transfection titer efficiency:Poor quality of Use the S.N.A.P. ™ pLenti6/V5 MidiPrep Kit to plasmidDNA prepare plasmid DNA. Unhealthy 293FT cells; Use healthy 293FT cellsexhibit cells; do not overgrow. low viability Cells should be 90-95%confluent at the time of transfection. 293FT cells Optimize such thatplated too plasmid DNA (in sparsely □g):Lipofectamine ™ Plasmid 2000 (in□1) ratio DNA:transfection ranges from 1:2 to 1:3. reagent ratioincorrect Viral supernatant Concentrate virus using too dilute anymethod of choice (Yee, 1999). Viral supernatant DO NOT freeze/thawfrozen and thawed viral supernatant more multiple times than 3 times.Poor choice of Use an adherent cell line titering cell line with thecharacteristics discussed herein. Gene of interest Viral titersgenerally is large decrease as the size of the insert increases; insertslarger than 6 kb are not recommended. Gene of interest Generation ofconstructs is toxic to cells containing activated oncogenes orpotentially harmful genes is not recommended. Polybrene ® not Transducethe lentiviral included during construct into cells in transduction thepresence of Polybrene ®. No colonies Too much blasticidin Determine thesensitivity obtained used for selection of the cell line to upontitering blasticidin by performing a kill curve experiment. Use theminimum concentra- tion of blasticidin required to kill the untransducedcell line. Viral stocks stored Aliquot and store stocks incorrectly at−80° C. Do not freeze/thaw more than 3 times. Polybrene ® not Transducethe lentiviral included during construct into cells in transduction thepresence of Polybrene ®. Titer Too little blasticidin Increase amount ofindeterminable; used for selection blasticidin used for cells confluentselection. Viral supernatant Titer lentivirus using not diluted a widerrange of 10-fold sufficiently serial dilutions (e.g. 10⁻² to 10⁻⁸).

[0843] Below are listed some potential problems and possible solutionsthat may help troubleshoot transduction and expression experiment.Problem Reason Solution No expression Promoter The lentiviral constructsilencing may integrate into a chromo- somal region that silences theCMV promoter controlling expression of the gene of interest. Screenseveral blasticidin-resistant clones and select the one thatdemonstrates the highest expression levels of the recombinant protein.Viral stocks Aliquot and store stocks stored at −80° C. Do not freeze/incorrectly thaw more than 3 times. Poor Poor transduction Transduce thelentiviral expression efficiency: construct into cells in thePolybrene ® presence of Polybrene ®. not included Transduce thelentiviral during construct into cells using transduction a higher MOI.Non-dividing cell type used MOI too low Transduce the lentiviralconstruct into cells using a higher MOI. Too much Determine thesensitivity of blasticidin used the cell line to blasticidin forselection by performing a kill curve experiment. Use the minimumconcen-tration of blasticidin required to kill the untrans- duced cellline. Cells harvested Do not harvest cells until too soon after at least24-48 hours after transduction transduction to allow reversetranscription and integration of the lentivirus into the genome. Gene ofinterest Generation of constructs is toxic to cells containing activatedoncogenes or potentially harmful genes is not recommended.

[0844] Table 25 provides some of the characteristics of the vector pLP1.The complete sequence is provided as table 21. A plasmid map is providedas FIG. 37A. TABLE 25 Feature Benefit Human cytomegalovirus Permitshigh-level expression of (CMV) promoter the HIV-1 gag and pol genes inbases 1-747, mammalian cells (Andersson et al., TATA box bases 1989;Boshart et al., 1985; 648-651 Nelson et al., 1987). Human β-globinEnhances expression of the gag intron and pol genes in mammalian cells.bases 880-1320 HIV-1 gag coding Encodes the viral core proteins sequencerequired for forming the structure bases 1355-2857 of the lentivirus(Luciw, 1996). HIV-1 pol coding Encodes the viral replication sequenceenzymes required for replication bases 2650-5661 and integration of thelenti- virus (Luciw, 1996). HIV-1 Rev response Permits Rev-dependentexpression element (RRE) of the gag and pol genes bases 5686-5919 Humanβ-globin poly- Allows efficient transcription adenylation signaltermination and polyadenylation bases 6072-6837 of mRNA. pUC origin ofrepli- Permits high-copy replication cation (ori) and maintenance in E.coli. bases 6995-7668 complementary strand Ampicillin (bla) Allowsselection of the plasmid resistance gene in E. coli. bases 7813-8673complementary strand bla promoter bases 8674-8772 complementary strand

[0845] Table 26 provides some of the characteristics of the vector pLP2.The complete sequence is provided as Table 22. A plasmid map is providedas FIG. 37B. TABLE 26 Feature Benefit RSV enhancer/promoter Permitshigh-level expression bases 1-271, TATA box of the rev gene (Gorman etal., bases 200-207, 1982). transcription initiation base 229 RSV UTRbases 230-271 HIV-1 Rev ORF Encodes the Rev protein which bases 391-741interacts with the RRE on pLP1 to induce Gag and Pol expression, and onthe pLenti6/V5 expression vector to promote the nuclear export of theunspliced viral RNA for packaging into viral particles. HIV-1 LTRpolyadenylation Allows efficient transcription signal termination andpolyadenylation bases 850-971 of mRNA. Ampicillin (bla) Allows selectionof the plasmid resistance gene in E. coli. promoter bases 1916-2014 genebases 2015-2875 pUC origin of replication Permits high-copy replication(ori) and maintenance in E. coli. bases 3020-3693

[0846] Table 27 provides some of the characteristics of the vectorpLP/VSVG. The complete sequence is provided as Table 23. A plasmid mapis provided as FIG. 37C. TABLE 27 Feature Benefit Human CMV promoterPermits high-level expression bases 1-747 of the VSV-G gene in mammaliancells (Andersson et al., 1989; Boshart et al., 1985; Nelson et al.,1987). Human (β-globin intron Enhances expression of the bases 880-1320VSV-G gene in mammalian cells. VSV G glycoprotein (VSV-G) Encodes theenvelope G glyco- bases 1346-2881 protein from Vesicular StomatitisVirus to allow production of a pseudotyped retrovirus with a broad hostrange (Burns et al., 1993; Emi et al., 1991; Yee et al., 1994). Humanβ-globin Allows efficient transcription polyadenylation signaltermination and polyadenylation bases 3004-3769 of mRNA. pUC origin ofreplication Permits high-copy replication (ori) and maintenance in E.coli. bases 3927-4600 complementary strand Ampicillin (bla) resistanceAllows selection of the plasmid gene in E. coli. gene bases 4745-5606complementary strand promoter bases 5606-5704 complementary strand

Example 11 GATEWAY™-Adapted Destination Vector for Cloning andHigh-Level Expression in Mammalian Cells Using the ViraPower™ LentiviralExpression System

[0847] ViraPower™ Lentiviral Expression Products

[0848] The pLenti6/V5-DEST , pLenti4/V5-DEST, and pLenti6/UbC/V5-DESTvectors are designed for use with the ViraPower™ Lentiviral ExpressionSystem available from Invitrogen Corporation, Carlsbad, Calif., which isdiscussed in some detail above. Depending on the vector chosen, thepLenti-DEST vectors are available with the human cytomegalovirus (CMV)immediate early promoter or the human ubiquitin C (UbC) promoter tocontrol expression of the gene of interest, and the Zeocin™ resistancegene or the blasticidin resistance gene for selection in E. coli ormammalian cells.

[0849] Expression of a recombinant fusion protein can be detected usingan antibody to the V5 epitope. Horseradish peroxidase (HRP) or alkalinephosphatase (AP)-conjugated antibodies allow one-step detection usingchemiluminescent or colorimetric detection methods. A fluoresceinisothiocyanate (FITC)-conjugated antibody allows one-step detection inimmunofluorescence experiments. Suitable detection reagents for fusionproteins can be obtained from Invitrogen Corporation, Carlsbad, Calif.,for example, Anti-V5 Antibody, catalog number R960-25, Anti-V5-HRPAntibody, catalog number R961-25, Anti-V5-AP Antibody, catalog numberR962-25, Anti-V5-FITC Antibody, catalog number R963-25.

[0850] pLenti6/V5-DEST™ is an 8.7 kb vector adapted for use with theGATEWAY™ Technology, and is designed to allow high-level expression ofrecombinant fusion proteins in dividing and non-dividing mammalian cellsusing Invitrogen's ViraPower™ Lentiviral Expression System. A map of thevector is provided as FIG. 36A and the sequence of the vector isprovided as Table 17.

[0851] The pLenti-DEST vectors contain the following features: RousSarcoma Virus (RSV) enhancer/promoter for Tat-independent production ofviral mRNA in the producer cell line (Dull et al., 1998); modified HIV-15′ and 3′ Long Terminal Repeats (LTR) for viral packaging and reversetranscription of the viral mRNA (Dull et al., 1998; Luciw, 1996) (Note:The U3 region of the 3′ LTR is deleted (ΔU3) and facilitatesself-inactivation of the 5′ LTR after transduction to enhance thebiosafety of the vector (Dull et al., 1998)); HIV-1 psi (Ψ) packagingsequence for viral packaging (Luciw, 1996); HIV Rev response element(RRE) for Rev-dependent nuclear export of unspliced viral mRNA (Kjems etal., 1991, Proc. Natl. Acad. Sci. USA 88, 683-687; Malim et al., 1989,Nature 338, 254-257); human CMV or UbC promoter for constitutiveexpression of the gene of interest from a viral or cellular promoter,respectively; two recombination sites, attR1 and attR2, downstream ofthe CMV or UbC promoter for recombinational cloning of the gene ofinterest from an entry clone; chloramphenicol resistance gene (Cm^(R))located between the two attR sites for counterselection; the ccdB genelocated between the attR sites for negative selection; C-terminal V5epitope for detection of the recombinant protein of interest (Southernet al., 1991, J. Gen. Virol. 72, 1551-1557); blasticidin (Izumi et al.,1991; Kimura et al., 1994; Takeuchi et al., 1958; Yamaguchi et al.,1965) or Zeocin™ (Drocourt et al., 1990, Nucleic Acids Res. 18, 4009;Mulsant et al., 1988, Somat. Cell Mol. Genet. 14, 243-252) resistancegene for selection in E. coli and mammalian cells; ampicillin resistancegene for selection in E. coli; and the pUC origin for high-copyreplication of the plasmid in E. coli.

[0852] A control plasmid containing the lacZ gene is included with eachpLenti-DEST vector for use as a positive expression control in themammalian cell line of choice.

[0853] The pLenti4/V5-DEST and pLenti6/V5-DEST vectors use the human CMVimmediate early promoter to allow high-level, constitutive expression ofthe gene of interest in mammalian cells (Andersson et al., 1989; Boshartet al., 1985; Nelson et al., 1987). The sequence of the pLenti4/V5-DESTplasmid is provided as Table 19. Although highly active in mostmammalian cell lines, activity of the viral CMV promoter can bedown-regulated in some cell lines due to methylation (Curradi et al.,2002, Mol. Cell. Biol. 22, 3157-3173), histone deacetylation (Rietveldet al., 2002, EMBO J. 21, 1389-1397), or both.

[0854] The pLenti6/UbC/V5-DEST vector uses the human UbC promoter toallow constitutive, but more physiological levels of expression from thegene of interest in mammalian cells (Marinovic et al., 2000, Biophys.Res. Comm. 274, 537-541). The sequence of the pLenti6/UbC/V5-DESTplasmid is provided as Table 20. When compared to the CMV promoter, theUbC promoter is generally 2-4 fold less active. The UbC promoter is notdown-regulated, making it useful for transgenic studies (Gill et al.,2001, Gene Ther. 8, 1539-1546; Lois et al., 2002, Science 295, 868-872;Marinovic et al., 2000; Schorpp et al., 1996, Nuc. Acids Res. 24,1787-1788; Yew et al., 2001, Mol. Ther. 4, 75-82). The human ubiquitin C(UbC) promoter (in pLenti6/UbC/V5-DEST) allows high-level expression ofrecombinant protein is most mammalian cell lines (Wulffet al., 1990,FEBS Lett. 261, 101-105) and in virtually all tissues tested intransgenic mice (Schorpp et al., 1996). The diagram below shows thefeatures of the UbC promoter as described by Nenoi et al., 1996 Gene175, 179-185.

[0855] GATEWAY™ is a universal cloning technology that takes advantageof the site-specific recombination properties of bacteriophage lambda(Landy, 1989) to provide a rapid and highly efficient way to move a geneof interest into multiple vector systems. To express a sequence ofinterest (e.g., a sequence encoding a polypeptide of interest) inmammalian cells using the GATEWAY™ technology, simply: clone thesequence of interest into a GATEWAY™ entry vector of choice to create anentry clone; generate an expression clone by performing an LRrecombination reaction between the entry clone and a GATEWAY™destination vector (e.g. pLenti4/V5-DEST, pLenti6/V5-DEST, orpLenti6/UbC/V5-DEST); and use the expression clone in the ViraPower™Lentiviral Expression System.

[0856] For more detailed information about GATEWAY™ System, generatingan entry clone, and performing the LR recombination reaction, refer tothe GATEWAY™ Technology manual available from Invitrogen Corporation,Carlsbad, Calif.

[0857] The pLenti4/V5-DEST, pLenti6/V5-DEST, and pLenti6/UbC/V5-DESTvectors are supplied as supercoiled plasmids. Although the GATEWAY™Technology Manual has previously recommended using a linearizeddestination vector for more efficient recombination, further testing atInvitrogen has found that linearization of pLenti6/V5-DEST™ is notrequired to obtain optimal results for any downstream application.

[0858] To propagate and maintain the pLenti4/V5-DEST, pLenti6/V5-DEST,or pLenti6/UbC/V5-DEST vectors, Library Efficiency® DB3.1™ CompetentCells (Catalog no. 11782-018) from Invitrogen Corporation, Carlsbad,Calif. are recommended for transformation. The DB3.1™ E. coli strain isresistant to CcdB effects and can support the propagation of plasmidscontaining the ccdB gene. To maintain integrity of the vector, selectfor transformants in media containing 50-100 μg/ml ampicillin and 15μg/ml chloramphenicol. In one alternative of this aspect of theinvention, the chloramphenicol resistance gene in the cassette can bereplaced by a spectinomycin resistance gene (see Hollingshead et al.,Plasmid 13(1):17-30 (1985), NCBI accession no. X02340 M10241), and thedestination vector containing attP sites flanking the ccdB andspectinomycin resistance genes can be selected onampicillin/spectinomycin-containing media. It has recently been foundthat the use of spectinomycin selection instead of chloramphenicolselection results in an increase in the number of colonies obtained onselection plates, indicating that use of the spectinomycin resistancegene may lead to an increased efficiency of cloning from that observedusing cassettes containing the chloramphenicol resistance gene. Do notuse general E. coli cloning strains including TOP10 or DH5α forpropagation and maintenance as these strains are sensitive to CcdBeffects.

[0859] To recombine a sequence of interest into pLenti4/V5-DEST,pLenti6/V5-DEST, or pLenti6/UbC/V5-DEST, an entry clone containing thesequence must be created. Many entry vectors including pENTR/D-TOPO® areavailable from Invitrogen Corporation, Carlsbad, Calif. to facilitategeneration of entry clones.

[0860] pLenti4/V5-DEST, pLenti6/V5-DEST, and pLenti6/UbC/V5-DEST areC-terminal fusion vectors. To express a fusion polypeptide of apolypeptide encoded by a sequence of interest with the V5 epitope codingsequence present in the vector, a sequence of interest must contain anATG initiation codon in the context of a Kozak translation initiationsequence for proper initiation of translation in mammalian cells (Kozak,1987; Kozak, 1991; Kozak, 1990). An example of a Kozak consensussequence is (G/A)NNATGG. Other sequences are possible, but the G or A atposition −3 and the G at position +4 are the most critical for function(shown in bold). The ATG initiation codon is underlined. The readingframe of the polypeptide encoded by the sequence of interest must be inframe with the C-terminal tag containing the V5 epitope afterrecombination and the sequence of interest must not contain a stop codonin this reading frame. The C-terminal peptide containing the V5 epitopeand the attB2 site will add approximately 4.5 kDa to the size of thepolypeptide encoded by the sequence of interest.

[0861] Each entry clone contains attL sites flanking the gene ofinterest. Genes in an entry clone are transferred to the destinationvector backbone by mixing the DNAs with the GATEWAY™ LR Clonase™ EnzymeMix available from Invitrogen Corporation, Carlsbad, Calif. Theresulting recombination reaction is then transformed into E. coli (e.g.TOP10 or DH5α™-T1^(R)) and the expression clone selected (e.g., usingampicillin and blasticidin). Recombination between the attR sites on thedestination vector and the attL sites on the entry clone replaces thechloramphenicol (Cm^(R)) gene and the ccdB gene with the gene ofinterest and results in the formation of attB sites in the expressionclone.

[0862] Any recA, endA E. coli strain including TOP10, DH5α™, orequivalent may be used for transformation. Do not transform the LRreaction mixture into E. coli strains that contain the F′ episome (e.g.TOP10F′). These strains contain the ccdA gene and will prevent negativeselection with the ccdB gene.

[0863] When transforming E. coli with the recombination reaction(pLenti4/V5-DEST, pLenti6/V5-DEST, or pLenti6/UbC/V5-DEST× entry clone),unwanted recombination (less than 5%) between the 5′ and 3′ LTRs hasbeen observed when transformants are selected on LB agar platescontaining ampicillin. These events occur less frequently when selectionis performed using 100 μg/ml ampicillin and an additional selection, forexample, 50 μg/ml blasticidin for pLenti6/V5-DEST or pLenti6/UbC/V5-DESTor 25 μg/ml Zeocin™ for pLenti4/V5-DEST. For Zeocin™ to be active, thesalt concentration of the bacterial medium must be <90 mM and the pHmust be 7.5. Therefore, selection on LB agar plates containing 50-100μg/ml ampicillin and an additiona selection agent is recommended. Notethat transformed E. coli grow more slowly in LB media containingampicillin and blasticidin, and may require slightly longer incubationtimes to obtain visible colonies. Transformants that contain arecombined plasmid generally give rise to larger colonies than thosecontaining an intact plasmid.

[0864] The ccdB gene mutates at a very low frequency, resulting in avery low number of false positives. True expression clones will bechloramphenicol-sensitive and ampicillin- and blasticidin-resistant (forpLenti6 vectors) and ampicillin- and Zeocin™-resistant (forpLenti4/V5-DEST). Transformants containing a plasmid with a mutated ccdBgene will be ampicillin-, blasticidin- or Zeocin™-, andchloramphenicol-resistant, as appropriate. To check a putativeexpression clone, test for growth on LB plates containing 30 μg/mlchloramphenicol. A true expression clone should not grow in the presenceof chloramphenicol.

[0865]FIG. 46A provides a diagram of the recombination region ofpLenti6/V5-DEST™ or pLenti4/V5-DEST after a recombination reaction witha sequence of interest. Shaded regions correspond to the sequence ofinterest transferred from the entry clone into the pLenti6/V5-DEST™vector by recombination. Non-shaded regions are derived from thepLenti6/V5-DEST™ or pLenti4/V5-DEST vector. Bases 2448 and 4130 of thepLenti4/V5-DEST and pLenti6/V5-DEST™ sequences are marked. Restrictionssites are labeled to indicate the actual cleavage site.

[0866]FIG. 46B shows the recombination region of the expression cloneresulting from pLenti6/UbC/V5-DEST× entry clone. Note that this diagramdoes not contain the complete sequence of the UbC promoter. For adiagram of the UbC promoter see FIG. 46C. Shaded regions in FIG. 46Bcorrespond to those DNA sequences transferred from the entry clone intothe pLenti6/UbC/V5-DEST vector by recombination. Non-shaded regions arederived from the pLenti6/UbC/V5-DEST vector. Bases 3079 and 4762 of thepLenti6/UbC/V5-DEST sequence are marked.

[0867] Once an expression clone has been generated in thepLenti6/V5-DEST backbone, maintain and propagate the plasmid in LBmedium containing 50-100 μg/ml ampicillin. Addition of blasticidin isnot required.

[0868] To confirm that a gene of interest is in frame with theC-terminal tag, sequence the expression construct, if desired. Refer toFIG. 46 for the location of the recommended primer binding sites (CMV orUbC forward priming site and V5(C-term) reverse priming site) to use tosequence the expression construct. To sequence a pLenti4/V5-DEST orpLenti6/V5-DEST construct, the CMV forward primer5′-CGCAAATGGGCGGTAGGCGTG-3′ and V5(C-term) reverse primer5′-ACCGAGGAGAGGGTTAGGGAT-3′ can be used. To sequence apLenti6/UbC/V5-DEST construct, the UB forward primer5′-TCAGTGTTAGACTAGTAAATTG-3′ and the V5(C-term) reverse primer5′-ACCGAGGAGAGGGTTAGGGAT-3′ can be used.

[0869] Once purified plasmid DNA of the expression construct has beenobtained, a viral stock can be prepared and used to transduce a cellline of choice as described above. Host cells containing the expressionclone can be propagated in LB medium with ampicillin. It is notnecessary to add an additional selection agent.

[0870] High salt and acidity or basicity inactivate Zeocin™. Therefore,it is recommended that the salt in bacterial medium be reduced and thepH adjusted to 7.5 to keep the drug active. Note that the pH and saltconcentration do not need to be adjusted when preparing tissue culturemedium containing Zeocin™. Store Zeocin™ at −20° C. and thaw on icebefore use. Zeocin™ is light sensitive. Store the drug, and plates ormedium containing drug, in the dark at +4° C. Culture medium containingZeocin™ may be stored at +4° C. protected from exposure to light for upto 1 month. Wear gloves, a laboratory coat, and safety glasses orgoggles when handling Zeocin™-containing solutions. Zeocin™ is toxic. Donot ingest or inhale solutions containing the drug.

[0871] The pLenti6/V5-DEST™ vector (8688 bp) contains the followingfeatures at the indicated locations. The locations of the features inthe pLenti6/V5-DEST plasmid are as follows: RSV/5′ LTR hybrid promoterbases 1-410; RSV promoter bases 1-229; HIV-1 5′ LTR bases 230-410; 5′splice donor base 520; HIV-1 psi (ψ) packaging signal bases 521-565;HIV-1 Rev response element (RRE) bases 1075-1308; 3′ splice acceptorbase 1656; 3′ splice acceptor base 1684; CMV promoter bases 1809-2392;attR1 site: bases 2440-2564; Chloramphenicol resistance gene (Cm^(R))bases 2673-3332; ccdB gene bases 3674-3979; attR2 site bases 4020-4144;V5 epitope bases 4197-4238; SV40 early promoter and origin bases4293-4602; EM7 promoter bases 4657-4723; Blasticidin resistance genebases 4724-5122; ΔU3/3′ LTR bases 5208-5442; ΔU3 bases 5208-5261; 3′LTR: bases 5262-5442; SV40 polyadenylation signal bases 5514-5645; blapromoter bases 6504-6602; Ampicillin (bla) resistance gene bases6603-7463; and pUC origin bases 7608-8281.

[0872] The pLenti4/V5-DEST vector(8634 nucleotides) contains thefollowing features at the indicated locations: RSV/5′ LTR hybridpromoter bases 1-410; RSV promoter bases 1-229; HIV-1 5′ LTR bases230-410; 5′ splice donor base 520; HIV-1 psi (ψ) packaging signal bases521-565; HIV-1 Rev response element (RRE) bases 1075-1308; 3′ spliceacceptor base 1656; 3′ splice acceptor base 1684; CMV promoter bases1809-2392; attR1 site bases 2440-2564; Chloramphenicol resistance gene(Cm^(R)) bases 2673-3332; ccdB gene bases 3674-3979; attR2 site bases4020-4144; V5 epitope bases 4197-4238; SV40 early promoter and originbases 4293-4602; EM7 promoter bases 4621-4687; Zeocin™ resistance genebases 4688-5062; ΔU3/3′ LTR bases 5154-5388; ΔU3 bases 5154-5207; 3′ LTRbases 5208-5388; SV40 polyadenylation signal bases 5460-5591; blapromoter bases 6450-6548; Ampicillin (bla) resistance gene bases6549-7409; and the pUC origin bases 7554-8227.

[0873] The pLenti6/UbC/V5-DEST vector (9320 nucleotides) contains thefollowing features at the indicated locations: RSV/5′ LTR hybridpromoter bases 1-410; RSV promoter bases 1-229; HIV-1 5′ LTR bases230-410; 5′ splice donor base 520; HIV-1 psi (ψ) packaging signal bases521-565; HIV-1 Rev response element (RRE) bases 1075-1308; 3′ spliceacceptor base 1656; 3′ splice acceptor base 1684; UbC promoter bases1798-3016; attR1 site bases 3072-3196; Chloramphenicol resistance gene(Cm^(R)) bases 3305-3964; ccdB gene bases 4306-4611; attR2 site bases4652-4776; V5 epitope bases 4829-4870; SV40 early promoter and originbases 4925-5234; EM7 promoter bases 5289-5355; Blasticidin resistancegene bases 5356-5754; ΔU3/3′ LTR bases 5840-6074; ΔU3 bases 5840-5893;3′ LTR bases 5894-6074; SV40 polyadenylation signal bases 6146-6277; blapromoter bases 7136-7234; Ampicillin (bla) resistance gene bases7235-8095; and the pUC origin bases 8240-8913.

Example 12 Five-Minute, Directional TOPO® Cloning of Blunt-End PCRProducts into an Expression Vector for the ViraPower™ LentiviralExpression System

[0874] The following protocol may be used to clone a nucleic acidsegment using topoisomerase. Other protocols known to those skilled inthe art are also suitable. An example of another suitable protocol maybe found in the pENTR Directional TOPO® Cloning Kit manual availablefrom Invitrogen Corporation, Carlsbad, Calif. (catalog number 25-0434).Step Action Design PCR Primers Include the 4 base pair sequences (CACC)necessary for directional cloning on the 5′ end of the forward primer.Design the primers such that a gene of interest will be optimallyexpressed and fused in frame with the V5 epitope tag, if desired.Amplify the Gene of Use a thermostable, proofreading DNA Interestpolymerase and the PCR primers above to produce blunt-end PCR product.Use agarose gel electrophoresis to check the integrity of PCR product.Perform the TOPO ® 1. Set up the following TOPO ® Cloning ReactionCloning reaction. Fresh PCR product 0.5 to 4 μl Salt Solution 1 μlSterile water add to a final volume of 5 μl TOPO ® vector 1 μl Totalvolume 6 μl 2. Mix gently and incubate for 5 minutes at roomtemperature. 3. Place on ice and proceed to trans- form One Shot ® TOP10 chemically competent E. coli, below. Transform One Shot ® 1. Add 2 μlof the TOPO ® Cloning TOP 10 Chemically reaction into a vial of OneShot ® Competent E. coli TOP 10 chemically competent E. coli and mixgently. 2. Incubate on ice for 5 to 30 minutes. 3. Heat-shock the cellsfor 30 seconds at 42° C. without shaking. Immediately transfer the tubeto ice. 4. Add 250 μl of room temperature SOC medium. 5. Incubate at 37°C. for 1 hour with shaking. 6. Spread 50-200 μl of bacterial culture ona prewarmed LB agar plate containing 50-100 μg/ml ampi- cillin and 50μg/ml blasticidin, and incubate overnight at 37° C.

[0875] Using the Control PCR Template and the Control PCR Primersincluded with the kit to perform a control reaction is recommended. Seethe protocol below for details.

[0876] The pLenti6/V5 Directional TOPO® Cloning Kit is shipped on dryice and contains two boxes. Upon receipt, store the boxes as detailedbelow. Box Item Storage 1 pLenti6/V5-D-TOPO ® Reagents −20° C. 2 OneShot ® TOP 10 Chemically Competent E. coli −80° C.

[0877] pLenti6/V5-D-TOPO® reagents (Box 1) are listed below. Note thatthe user must supply a thermostable, proofreading polymerase and theappropriate PCR buffer. Item Concentration Amount pLenti6/V5-D-TOPO ® 10ng/μl linearized plasmid DNA in: 20 μl 50% glycerol 50 mM Tris-HCl, pH7.4 (at 25° C.) 1 mM EDTA 2 mM DTT 0.1% Triton X-100 100 μg/ml BSA 30 μMbromophenol blue dNTP Mix 12.5 mM dATP 10 μl 12.5 mM dCTP 12.5 mM dGTP12.5 mM dTTP in water, pH 8 Salt Solution 1.2 M NaCl 50 μl 0.06 M MgCl2Sterile Water — 1 ml CMV Forward 0.1 μg/μl in TE Buffer, pH 8 20 μlSequencing Primer V5(C-term) Reverse 0.1 μg/μl in TE Buffer, pH 8 20 μlSequencing Primer Control PCR Primers 0.1 μg/μl each in TE Buffer, pH 810 μl Control PCR Template 0.1 μg/μl in TE Buffer, pH 8 10 μlpLenti6/V5-GW/lacZ Lyophilized in TE Buffer, pH 8 10 μg ExpressionControl Plasmid

[0878] The sequences of CMV Forward and V5(C-term) Reverse sequencingprimers. Two micrograms of each primer are as follows: CMV Forward5′-CGCAAATGGGCGGTAGGCGTG-3′ V5(C-term) Reverse5′-ACCGAGGAGAGGGTTAGGGAT-3′

[0879] TOP10 cells have the following genotype: F⁻ mcrAΔ(mrr-hsdRMS-mcrBC) Φ80lacZΔM15 ΔlacX74 recA1 deoR araD139Δ(ara-leu)7697 galU galK rpsL (Str^(R)) endA1 nupG. Transformationefficiency is 1×10⁹ cfu/μg DNA and they should be stored at −80° C.

[0880] The pLenti6/V5-D-TOPO® vector is designed for use with theViraPower™ Lentiviral Expression System available from InvitrogenCorporation, Carlsbad, Calif. Ordering information for the ViraPower™Lentiviral Expression System and other ViraPower™ lentiviral supportproducts and expression vectors is provided below. For more information,see the Invitrogen Corporation, Carlsbad, Calif. Web site. Item QuantityCatalog no. ViraPower ™ Lentiviral Directional 1 kit K4950-00 TOPO ®Expression Kit (includes ViraPower Lentiviral Support Kit and the 293FTCell Line) ViraPower ™ Lentiviral GATEWAY ™ 1 kit K4960-00 ExpressionKit pLenti6/V5-DEST ™ GATEWAY ™ 6 μg V496-10 Vector Pack ViraPowerLentiviral Support Kit 20 reactions K4970-00 (includes ViraPowerPackaging Mix, Lipofectamine ™ 2000, and blasticidin) 293FT Cell Line 3× 10⁶ cells R700-07

[0881] Some of the reagents supplied in the pLenti6/V5 Directional TOPO®Cloning Kit as well as other reagents suitable for use with the kit areavailable separately from Invitrogen Corporation, Carlsbad, Calif.Ordering information for these reagents is provided below. Item QuantityCatalog no. One Shot ® TOP 10 Chemically 10 reactions C4040-10 CompetentCells 20 reactions C4040-03 One Shot ® TOP 10 Electro- 10 reactionsC4040-50 competent Cells Ampicillin 200 mg 11593-019 Blasticidin 50 mgR210-01 ThermalAce ™ DNA Polymerase 200 units E0200 1000 units E1000Platinum ® Pfx DNA Polymerase 100 units 11708-013 Lipofectamine ™ 20000.75 ml 11668-027 1.5 ml 11668-019

[0882] The pLenti6/V5 Directional TOPO® Cloning Kit combines theViraPower™ Lentiviral Expression System with TOPO® Cloning technology toprovide a highly efficient, rapid cloning strategy for insertion ofblunt-end PCR products into a vector for expression in dividing andnon-dividing mammalian cells. TOPO® Cloning requires no ligase, post-PCRprocedures, or restriction enzymes.

[0883] pLenti6/V5-D-TOPO® is a 7.0 kb expression vector designed tofacilitate rapid, directional TOPO® Cloning and high-level expression ofPCR products in mammalian cells using the ViraPower™ LentiviralExpression System (Catalog nos. K4950-00) available from InvitrogenCorporation, Carlsbad, Calif. Features of the vector include: RSVenhancer/promoter bases 1-229; HIV-1 5′ LTR bases 230-410; 5′ splicedonor base 520; HIV-1 psi (ψ) packaging sequence bases 521-565; HIV-1Rev response element (RRE) bases 1075-1308; 3′ splice acceptor base1656; 3′ splice acceptor base 1684; CMV promoter bases 1809-2392; CMVforward priming site bases 2274-2294; directional TOPO® site bases2431-2444; V5 epitope bases 2473-2514; V5(C-term) reverse priming sitebases 2482-2502; SV40 early promoter and origin bases 2569-2878; EM7promoter bases 2933-2999; Blasticidin resistance gene bases 3000-3398;ΔU3/HIV-1 3′ LTR bases 3485-3718; ΔU3: bases 3485-3537; Truncated HIV-13′ LTR bases 3538-3718; SV40 polyadenylation signal bases 3790-3921; blapromoter: bases 4780-4878; ampicillin (bla) resistance gene bases4879-5739; and the pUC origin: bases 5884-6557.

[0884] The control plasmid, pLenti6/V5-GW/lacZ, may be used as apositive expression control in the mammalian cell line of choice.

[0885] The ViraPower™ Lentiviral Expression System facilitates highlyefficient, in vitro or in vivo delivery of a target gene to dividing andnon-dividing mammalian cells using a replication-incompetent lentivirus.Based on the lentikat™ system developed by Cell Genesys (Dull et al.,1998), the ViraPower™ Lentiviral Expression System possesses featureswhich enhance its biosafety while allowing high-level gene expression ina wider range of cell types than traditional retroviral systems. Toexpress a gene of interest in mammalian cells using the ViraPower™Lentiviral Expression System:

[0886] 1. TOPO® Clone a gene of interest into pLenti6/V5-D-TOPO® tocreate an expression construct.

[0887] 2. Cotransfect the pLenti6/V5-D-TOPO® expression plasmid and theViraPower™ Packaging Mix into the 293FT cell line to produce lentivirus.

[0888] 3. Use the lentiviral stock to transduce the mammalian cell lineof choice.

[0889] 4. Assay for “transient” expression of the recombinant protein orgenerate a stable cell line using blasticidin selection.

[0890] Detailed protocols for creating recombinant lentiviruses areknown (e.g., ViraPower™ Lentiviral Expression System manual, catalognos. K4950-00, K4960-00, K4970-00, K4975-00, K49580-00, K49585-00, andK49590-00, version D, Invitrogen Corporation, Carlsbad, Calif.).

[0891] Directional joining of double-strand DNA using TOPO®-chargedoligonucleotides occurs by adding a 3′ single-stranded end (overhang) tothe incoming DNA (Cheng and Shuman, 2000, Mol. Cell. Biol. 20,8059-8068.). This single-stranded overhang is identical to the 5′ end ofthe TOPO®-charged DNA fragment. The pLenti6/V5-D-TOPO® vector contains a4 nucleotide overhang sequence.

[0892] In this system, PCR products are directionally cloned by addingfour bases to the forward primer (CACC). The overhang in the cloningvector (GTGG) invades the 5′ end of the PCR product, anneals to theadded bases, and stabilizes the PCR product in the correct orientation.Inserts can be cloned in the correct orientation with efficiencies equalto or greater than 90%. A schematic representation of the process isshown in FIG. 47.

[0893] The design of the PCR primers to amplify a gene of interest iscritical for expression. Consider the following when designing PCRprimers: sequences required to facilitate directional cloning; sequencesrequired for proper translation initiation of the PCR product; andwhether or not a coding sequence contained by the PCR product is to befused in frame with the C-terminal V5 epitope tag.

[0894] When designing a forward PCR primer, consider the points below.

[0895] Refer to FIG. 48 for a diagram of the TOPO® Cloning site forpLenti6/V5-D-TOPO®.

[0896] To enable directional cloning, the forward PCR primer MUSTcontain the sequence, CACC, at the 5′ end of the primer. The 4nucleotides, CACC, base pair with the overhang sequence, GTGG, in thepLenti6/V5-D-TOPO® vector.

[0897] The sequence of interest should include a Kozak translationinitiation sequence with an ATG initiation codon for proper initiationof translation (Kozak, 1987; Kozak, 1991; Kozak, 1990). An example of aKozak consensus sequence is (G/A)NNATGG. Other sequences are possible,but the G or A at position −3 and the G at position +4 are the mostcritical for function (shown in bold). The ATG initiation codon isunderlined.

[0898] Below is the DNA sequence of the N-terminus of a theoreticalprotein and the proposed sequence for a forward PCR primer. The ATGinitiation codon is underlined.

[0899] If the forward PCR primer is designed as above, then the primerincludes the 4 nucleotides, CACC, required for directional cloning, andthe ATG initiation codon falls within the context of a Kozak sequence(see boxed sequence), allowing proper translation initiation of the PCRproduct in mammalian cells. The first three base pairs of the PCRproduct following the 5′CACC overhang will constitute a functionalcodon.

[0900] When designing a reverse PCR primer, consider the points below.Refer to FIG. 48 for a diagram of the TOPO® Cloning site forpLenti6/V5-D-TOPO®. To ensure that the PCR product clones directionallywith high efficiency, the reverse PCR primer should not be complementaryto the overhang sequence GTGG at the 5′ end. A one base pair mismatchcan reduce the directional cloning efficiency from 90% to 50%,increasing the likelihood of the PCR product cloning in the oppositeorientation (see below). Evidence of PCR products cloning in theopposite orientation from a two base pair mismatch has not beenobserved.

[0901] To fuse a PCR product in frame with the C-terminal tag containingthe V5 epitope, the reverse PCR primer can be designed to remove thenative stop codon in the gene of interest (see below). To produce anative C-terminal on an expressed polypeptide, include the nativesequence containing the stop codon in the reverse primer or make surethe stop codon is upstream from the reverse PCR primer binding site.

[0902] First Example of Reverse Primer Design. Below is the sequence ofthe C-terminus of a theoretical protein. The stop codon is underlined.DNA sequence: AAG TCG GAG CAC TCG ACG ACG GTG TAG-3′

[0903] To fuse the protein in frame with the C-terminal tag inpLenti6/V5-D-TOPO®, design the reverse PCR primer to start with thecodon just up-stream of the stop codon, but the last two codons containGTGG (underlined below), which is identical to the 4 bp overhangsequence. As a result, the reverse primer will be complementary to the 4bp overhang sequence, increasing the probability that the PCR productwill clone in the opposite orientation. This situation should beavoided. DNA sequence: AAG TCG GAG CAC TCG ACG ACG GTG TAG-3′ ProposedReverse PCR primer sequence: TG AGC TGC TGC CAC AAA-5′

[0904] Another solution is to design the reverse primer so that ithybridizes just down-stream of the stop codon, but still includes theC-terminus of the ORF. Note that the stop codon will need to be replacedby a codon for an innocuous amino acid such as glycine, alanine, orlysine.

[0905] Second Example of Reverse Primer Design

[0906] Below is the sequence for the C-terminus of a theoreticalprotein. The stop codon is underlined.

[0907] . . . GCG GTT AAG TCG GAG CAC TCG ACG ACT GCA TAG-3′

[0908] To fuse the ORF in frame with the C-terminal tag inpLenti6/V5-D-TOPO®, remove the stop codon by starting with nucleotideshomologous to the last codon (TGC) and continue upstream. The reverseprimer will be:

[0909] 5′-TGC AGT CGT CGA GTG CTC CGA CTT-3′

[0910] This will amplify the C-terminus without the stop codon and allowthe ORF to be joined in frame with the C-terminal tag. To avoid joiningthe ORF in frame with a C-terminal tag, design the reverse primer toinclude the stop codon.

[0911] 5′-CTA TGC AGT CGT CGA GTG CTC CGA CTT-3′

[0912] pLenti6/V5-D-TOPO® accepts blunt-end PCR products. Do not add 5′phosphates to primers for PCR. This will prevent ligation into thepLenti6/V5-D-TOPO® vector. It is recommended that oligonucleotides begel-purified, especially if they are long (>30 nucleotides). Note thatpLenti6/V5-D-TOPO® is supplied linearized with both ends adapted withtopoisomerase I (see FIG. 47). The sequence of pLenti6/V5-D-TOPO™ isprovided as Table 18.

[0913] Once a PCR strategy has been decided upon and primerssynthesized, a blunt-end PCR product can be produced using anythermostable, proof-reading polymerase including, but not limited to,ThermalAce™, Platinum® Pfx, Pfu, or Vent® for PCR.

[0914] Follow the manufacturer's instructions and recommendations toproduce blunt-end PCR products. It is important to optimize PCRconditions to produce a single, discrete PCR product. PCR fragments maybe gel purified using standard techniques.

[0915] It is recommended that a 7 to 30 minute final extension be usedin the PCR reaction to ensure that all PCR products are completelyextended.

[0916] After the PCR reaction, the PCR product should be checked byremoving 5 to 10 μl from each PCR reaction and using agarose gelelectrophoresis to verify the quality and quantity of the PCR product.Check for a single, discrete band of the correct size. If there is not asingle, discrete band, follow the manufacturer's recommendations foroptimizing PCR with the polymerase of choice. Alternatively, gel-purifythe desired product.

[0917] Estimate the concentration of the PCR product. A 5:1 molar ratioof PCR product:TOPO® vector is recommended to obtain the highest TOPO®Cloning efficiency (e.g. use 5-10 ng of a 1 kb PCR product or 10-20 ngof a 2 kb PCR product in a TOPO® Cloning reaction). Adjust theconcentration of the PCR product as necessary before proceeding to TOPO®Cloning. If ThermalAce™ polymerase is used to produce blunt-end PCRproduct, note that ThermalAce™ can generate higher yields than otherproofreading polymerases. When generating PCR products in the 0.5 to 1.0kb range, generally the PCR reaction can be diluted 1:5 in 1×ThermalAce™ buffer before performing the TOPO® Cloning reaction. For PCRproducts larger than 1.0 kb, dilution may not be required.

[0918] Including salt (250 mM NaCl, 10 mM MgCl₂) in the TOPO® Cloningreaction may result in an increase in the number of transformants.Therefore, it is recommended that salt be added to the TOPO® Cloningreaction. A stock salt solution is provided in the kit for this purpose.Note that the amount of salt added to the TOPO® Cloning reaction variesdepending on whether chemically competent cells (provided) orelectrocompetent cells are to be transformed. For this reason twodifferent TOPO® Cloning reactions are provided to obtain the bestpossible results.

[0919] Transforming Chemically Competent E. coli. For TOPO® Cloning andtransformation into chemically competent E. coli, adding sodium chlorideand magnesium chloride to a final concentration of 250 mM NaCl, 10 mMMgCl₂ in the TOPO® Cloning reaction increases the number of coloniesover time. A Salt Solution (1.2 M NaCl, 0.06 M MgCl₂) is provided toadjust the TOPO® Cloning reaction to the recommended concentration ofNaCl and MgCl₂.

[0920] Transforming Electrocompetent E. coli. For transformation ofelectrocompetent E. coli, the amount of salt in the TOPO® Cloningreaction should be reduced (e.g., to 50 mM NaCl, 2.5 mM MgCl₂) toprevent arcing. Dilute the Salt Solution 4-fold with water to prepare a300 mM NaCl, 15 mM MgCl₂ solution for convenient addition to the TOPO®Cloning reaction (see below).

[0921] Setting Up the TOPO® Cloning Reaction. The table below describeshow to set up a TOPO® Cloning reaction (6 μl) for eventualtransformation into either chemically competent One Shot® TOP10 E. coli(provided) or electrocompetent E. coli. Additional information onoptimizing the TOPO® Cloning reaction can be found herein. If the PCRproduct was generated using ThermalAce™ polymerase, note that it may benecessary to dilute the PCR reaction before proceeding. The blue colorof the TOPO® vector solution is normal and is used to visualize thesolution. Chemically Competent Electrocompetent Reagents* E. coli E.coli Fresh PCR product 0.5 to 4 μl 0.5 to 4 μl Salt Solution 1 μl —Sterile Water add to a final volume of add to a final volume of 5 μl 5μl TOPO ® vector 1 μl 1 μl

[0922] *Store all reagents at −20° C. when finished. Salt solution andwater can be stored at room temperature or +4° C.

[0923] Performing the TOPO® Cloning Reaction. Mix reaction gently andincubate for 5 minutes at room temperature (22-23° C.). For mostapplications, 5 minutes will yield plenty of colonies for analysis.Depending on needs, the length of the TOPO® Cloning reaction can bevaried from 30 seconds to 30 minutes. For routine subcloning of PCRproducts, 30 seconds may be sufficient. For large PCR products (>1 kb)or TOPO® Cloning a pool of PCR products, increasing the reaction timemay yield more colonies.

[0924] Place the reaction on ice and transform suitable host cells usingstandard protocols. The TOPO® Cloning reaction can be stored at −20° C.overnight.

[0925] Transforming One Shot® TOP10 Competent E. coli. Once the TOPO®Cloning reaction has been performed, the pLenti6/V5-D-TOPO® construct istransformed into competent E. coli. One Shot® TOP10 Chemically CompetentE. coli (Invitrogen Corporation, Carlsbad, Calif.) are included with thekit to facilitate transformation, however, electrocompetent cells mayalso be used. Protocols to transform chemically competent orelectrocompetent E. coli are known to those skilled in the art.pLenti6/V5-D-TOPO® contains the ampicillin and blasticidin resistancegenes for selection of transformants. Unwanted recombination (less than5%) between the 5′ and 3′ LTRs has been observed when transformants areselected on LB agar plates containing ampicillin. These events occurless frequently when transformants are selected on LB agar platescontaining ampicillin and blasticidin. Transformants should be selectedon LB agar plates containing 50-100 μg/ml ampicillin AND 50 μg/mlblasticidin. Note that transformed E. coli grow more slowly in LB mediacontaining ampicillin and blasticidin, and may require slightly longerincubation times to obtain visible colonies.

[0926] Transformants that contain a recombined plasmid generally giverise to larger colonies than those containing an intact plasmid. Thereis no blue-white screening for the presence of inserts. Mosttransformants will contain recombinant plasmid with the PCR product ofinterest cloned in the correct orientation. Sequencing primers areincluded in the kit to sequence across an insert in the multiple cloningsite to confirm orientation and reading frame.

[0927] Addition of the Dilute Salt Solution to the TOPO® CloningReaction brings the final concentration of NaCl and MgCl₂ in thereaction to 50 mM and 2.5 mM, respectively. To prevent arcing of samplesduring electroporation, the volume of cells should be between 50 and 80μl (0.1 cm cuvettes) or 100 to 200 μl (0.2 cm cuvettes). If arcingduring transformation is seen, try reducing the voltage normally used tocharge the electroporator by 10%, reducing the pulse length by reducingthe load resistance to 100 ohms, and/or ethanol precipitating the TOPO®Cloning reaction and resuspending in water prior to electroporation.

[0928] After transformation and plating, pick 5 colonies and culturethem overnight in LB or SOB medium containing 50-100 μg/ml ampicillin.Addition of blasticidin is not required. Isolate plasmid DNA using amethod of choice. If ultra-pure plasmid DNA is need for automated ormanual sequencing, the S.N.A.P.™ MidiPrep Kit (Invitrogen Corporation,Carlsbad, Calif. Catalog no. K1910-01) may be used. Analyze the plasmidsby restriction analysis to confirm the presence and correct orientationof the insert. Use a restriction enzyme or a combination of enzymes thatcut once in the vector and once in the insert.

[0929] Sequencing. The construct may be sequenced to confirm that thesequence of interest is cloned in the correct orientation and in framewith the V5 epitope. The CMV Forward and V5(C-term) Reverse primers areincluded in the kit and can be used to sequence the insert.

[0930] The sequence for pLenti6/V5-D-TOPO® shown in Table 18 includesthe overhang sequence (GTGG) hybridized to CACC.

[0931] Analyzing Transformants by PCR. Transformants can be analyzedusing PCR. For PCR primers, use a combination of the CMV Forward primeror the V5(C-term) Reverse primer and a primer that hybridizes within theinsert. Appropriate amplification conditions can be determined by oneskilled in the art. Results from the PCR reaction may be verified byconducting restriction analysis in parallel. Artifacts may be obtainedin the PCR reaction because of mispriming or contaminating template.

[0932] If transformants or the correct insert are not obtained, performthe control reactions described below.

[0933] Once the correct clone has been identified, a glycerol stock ofbacteria containing the plasmid may be prepared for long term storage.Also, a stock of plasmid DNA can be prepared and stored at −20° C.

[0934] Once a host cell containing a pLenti6/V5-D-TOPO® expressionplasmid has been prepared, maintain and propagate the plasmid in LBmedium containing 50-100 μg/ml ampicillin. Addition of blasticidin isnot required.

[0935] Optimizing the TOPO® Cloning Reaction. The high efficiency ofTOPO® Cloning allows the cloning process to be streamlined. To speed upthe process of cloning PCR products, the TOPO® Cloning reaction can beincubated for only 30 seconds instead of 5 minutes. Fewer transformantsmay be obtained; however, because of the high efficiency of TOPO®Cloning, most of the transformants will contain the insert. After adding2 μl of the TOPO® Cloning reaction to chemically competent cells,incubate on ice for only 5 minutes. Increasing the incubation time to 30minutes does not significantly improve transformation efficiency.

[0936] When TOPO® Cloning large PCR products, toxic genes, or cloning apool of PCR products, more transformants may be needed to obtain thedesired clones. To increase the number of colonies incubate thesalt-supplemented TOPO® Cloning reaction for 20 to 30 minutes instead of5 minutes. Increasing the incubation time of the salt-supplemented TOPO®Cloning reaction allows more molecules to ligate and may increase thetransformation efficiency. Addition of salt appears to preventtopoisomerase I from rebinding and nicking the DNA after it has ligatedthe PCR product and dissociated from the DNA.

[0937] To clone dilute PCR products, increase the amount of the PCRproduct, incubate the TOPO® Cloning reaction for 20 to 30 minutes,and/or concentrate the PCR product.

[0938] Once the sequence of interest has been TOPO® Cloned intopLenti6/V5-D-TOPO®, the ViraPower™ Lentiviral Expression System fromInvitrogen Corporation, Carlsbad, Calif. can be used to produce a viralstock, which may then be used to transduce a mammalian cell line ofchoice to express the recombinant protein (as described above).

Example 13 Growth and Maintenance of the 293FT Cell Line

[0939] The 293FT cell line may be transported using any technique knownto those skilled in the art, for example, by freezing the cells andtransporting them on dry ice. For long term storage, the cells may bestored in liquid nitrogen. The 293FT cell line is supplied as one vialcontaining 3×10⁶ frozen cells in 1 ml of Freezing Medium.

[0940] The 293FT cell line is genetically modified and carries thepUC-derived plasmid, pCMVSPORT6TAg.neo. A map of the vector is providedas FIG. 49. The pCMVSPORT6TAg.neo plasmid is derived from pCMVSPORT6,which has been modified to include the neomycin resistance gene forstable selection in mammalian cells (Southern and Berg, 1982, J. Molec.Appl. Gen. 1, 327-339). Expression of the neomycin resistance gene iscontrolled by the SV40 early enhancer/promoter from which the SV40origin of replication has been removed. The plasmid also contains thegene encoding the SV40 large T antigen to facilitate optimal virusproduction (e.g. Invitrogen's ViraPower™ Lentiviral Expression System)and to permit episomal replication of plasmids containing the SV40 earlypromoter and origin. Expression of the SV40 large T antigen iscontrolled by the human cytomegalovirus (CMV) promoter.

[0941] The 293FT cell line is derived from the 293F cell line (seebelow) and stably expresses the SV40 large T antigen from thepCMVSPORT6TAg.neo plasmid. Expression of the SV40 large T antigen iscontrolled by the human cytomegalovirus (CMV) promoter and is high-leveland constitutive. For more information about pCMVSPORT6TAg.neo, seebelow.

[0942] Studies have demonstrated maximal virus production in human 293cells expressing SV40 large T antigen (Naldini et al., 1996), making the293FT cell line a particularly suitable host for generating lentiviralconstructs using the ViraPower™ Lentiviral Expression System availablefrom Invitrogen (Catalog nos. K4950-00 and K4960-00).

[0943] The 293 cell line is a permanent line established from primaryembryonal human kidney transformed with sheared human adenovirus type 5DNA (Graham et al., 1977; Harrison et al., 1977, Virology 77, 319-329).The E1A adenovirus gene is expressed in these cells and participates intransactivation of some viral promoters, allowing these cells to producevery high levels of protein. The 293-F cell line available fromInvitrogen Corporation, Carlsbad, Calif. (Catalog no. 11625) is afast-growing variant of the 293 cell line, and was originally obtainedfrom Robert Horlick at Pharmacopeia.

[0944] Antibiotic Resistance. 293FT cells stably express the neomycinresistance gene from pCMVSPORT6TAg.neo and should be maintained inmedium containing Geneticin® at the concentration listed below.Expression of the neomycin resistance gene in 293FT cells is controlledby the SV40 enhancer/promoter. Geneticin® is available separately fromInvitrogen Corporation, Carlsbad, Calif. (catalog number 11811-023).

[0945] Media for 293FT Cells. It is recommended that 293FT cells begrown in complete medium (D-MEM (high glucose), 10% fetal bovine serum(FBS), 2 mM L-glutamine, 1% Pen-Strep (optional)). For selection 500μg/ml Geneticin® should be included. For freezing, 90% complete mediumand 10% DMSO should be used. FBS does not need to be heat-inactivatedfor use with the 293FT cell line. 293FT cells should be maintained inmedium containing Geneticina at the concentration listed above. If cellsare split at a 1:5 to 1:10 dilution, they will generally reach 80-90%confluence in 3-4 days.

[0946] Follow the general guidelines below to grow and maintain 293FTcells. All solutions and equipment that come in contact with the cellsmust be sterile. Always use proper sterile technique and work in alaminar flow hood. Before starting experiments, be sure to have cellsestablished and also have some frozen stocks on hand. Early-passagecells are recommended for experiments. Upon receipt of the cells fromInvitrogen Corporation, Carlsbad, Calif., grow and freeze multiple vialsof the 293FT cell line to ensure that an adequate supply ofearly-passage cells is available.

[0947] For general maintenance of cells, pass 293FT cells when they are80-90% confluent (generally every 3-4 days). Avoid overgrowing cellsbefore passaging.

[0948] Use trypan blue exclusion to determine cell viability. Log phasecultures should be >90% viable.

[0949] When thawing or subculturing cells, transfer cells intopre-warmed medium.

[0950] Cells should be at the appropriate confluence and at greater than90% viability prior to transfection.

[0951] As with other human cell lines, when working with 293FT cells,handle as potentially biohazardous material under at least BiosafetyLevel 2 (BL-2) containment.

[0952] The following protocol is designed to thaw 293FT cells toinitiate cell culture. The 293FT cell line is supplied in a vialcontaining 3×10⁶ cells in 1 ml of Freezing Medium.

[0953] Remove the vial of cells from the liquid nitrogen and thawquickly in a 37° C. water bath. Just before the cells are completelythawed, decontaminate the outside of the vial with 70% ethanol, andtransfer the cells to a T-75 flask containing 12 ml of complete mediumwithout Geneticin®. Incubate the flask at 37° C. for 2-4 hours to allowthe cells to attach to the bottom of the flask. Aspirate off the mediumand replace with 12 ml of fresh, complete medium without Geneticin®.Incubate cells overnight at 37° C. The next day, aspirate off the mediumand replace with fresh, complete medium containing Geneticin® at therecommended concentration listed above. Incubate the cells and checkthem daily until the cells are 80-90% confluent (2-7 days).

[0954] Passaging Cells. When cells are ˜80-90% confluent, remove allmedium from the flask. Wash cells once with 10 ml PBS to remove excessmedium and serum. Serum contains inhibitors of trypsin. Add 5 ml oftrypsin/versene (EDTA) solution to the monolayer and incubate 1 to 5minutes at room temperature until cells detach. Check the cells under amicroscope and confirm that most of the cells have detached. If cellsare still attached, incubate a little longer until most of the cellshave detached. Add 5 ml of complete medium to stop trypsinization.Briefly pipette the solution up and down to break up clumps of cells.

[0955] To maintain cells in 75 cm² flasks, transfer 1 ml of the 10 mlcell suspension from above to a new 75 cm² flask and add 15 ml fresh,complete medium containing Geneticin®. To have the cells reachconfluency sooner, split the cells at a lower dilution (i.e. 1:4).

[0956] To expand cells into 175 cm² flasks, add 28 ml of fresh, completemedium containing Geneticin® to each of three 175 cm² flasks, thentransfer 2 ml of the cell suspension to each flask to obtain a totalvolume of 30 ml.

[0957] Incubate flasks in a humidified, 37° C., 5% CO₂ incubator.

[0958] Passage the cells as necessary to maintain or expand cells.

[0959] Freezing Cells. When freezing the 293FT cell line, it isrecommended that the cells be frozen at a density of at least 3×10⁶viable cells/ml. Use a freezing medium composed of 90% complete mediumand 10% DMSO. Complete medium is medium containing serum.

[0960] Preparing Freezing Medium. Freezing medium should be preparedimmediately before use. In a sterile, conical centrifuge tube, mixtogether 0.9 ml of fresh complete medium and 0.1 ml of DMSO for every 1ml needed. Place the tube on ice until use. Discard any remainingfreezing medium after use.

[0961] Freezing the Cells. Before starting, label cryovials and preparefreezing medium (see above). Keep the freezing medium on ice. To collectcells, count the cells prepared by trypsinization as described inPassaging the Cells above. Pellet cells at 250×g for 5 minutes in atable top centrifuge at room temperature and carefully aspirate off themedium. Resuspend the cells at a density of at least 3×10⁶ cells/ml inchilled freezing medium. Place vials in a microcentrifuge rack andaliquot 1 ml of the cell suspension into each cryovial. Freeze cells inan automated or manual, controlled-rate freezing apparatus followingstandard procedures. For ideal cryopreservation, the freezing rateshould be a decrease of 1° C. per minute. Transfer vials to liquidnitrogen for long-term storage.

[0962] The viability and recovery of frozen cells may be checked 24hours after storing cryovials in liquid nitrogen by following theprocedure outlined in Thawing above.

[0963] Transfecting Cells. The 293FT cell line is generally amenable totransfection using standard methods including calcium phosphateprecipitation (Chen and Okayama, 1987, Molec. Cell. Biol. 7, 2745-2752;Wigler et al., 1977, Cell 11, 223-232), lipid-mediated transfection(Felgner et al., 1989, Proc. West. Pharmacol. Soc. 32, 115-121; Felgnerand Ringold, 1989, Nature 337, 387-388), and electroporation (Chu etal., 1987, Nucleic Acids Res. 15, 1311-1326; Shigekawa and Dower, 1988,BioTechniques 6, 742-751). Typically cationic lipid-based transfectionreagents are used to transfect 293FT cells. Lipofectamine™ 2000(Invitrogen Corporation, Carlsbad, Calif. catalog number 11668-027) isrecommended, but other transfection reagents are suitable.

[0964] Transient Transfection. The 293FT cell line may be transientlytransfected with any plasmid. Make sure that cells are healthy at thetime of plating. Overgrowth of cells prior to passaging can compromisetheir transfection efficiency. On the day before transfection, platecells such that they will be approximately 60% confluent at the time oftransfection. If Lipofectamine™ 2000 is to be used as a transfectionreagent, plate cells such that they will be 90-95% confluent at the timeof transfection. Transfect the plasmid construct into the 293FT cellline using the method of choice (see above). After transfection, addfresh medium containing 500 μg/ml Geneticin® and allow the cells torecover for 24-48 hours before proceeding to assay for expression of thegene of interest.

[0965] Generating Stable Cell Lines. 293FT cells can be used as hosts togenerate a stable cell line expressing a gene of interest from mostplasmids.

[0966] Remember that the introduced plasmid must contain a selectionmarker other than neomycin resistance. Stable cell lines can then begenerated by transfection and dual selection with Geneticin® and theappropriate selection agent.

[0967] Since 293FT cells stably express the SV40 large T antigen,generating stable cell lines with plasmids that contain the SV40 originof replication is not recommended.

Example 14 Use of Suppressor tRNAs to Transiently Label Proteins ofInterest

[0968] This example describes the use of mammalian suppressor tRNAs(e.g., tRNA^(ser)) that specifically recognize and decode one of thethree stop codons: amber (TAG), opal (TGA) or ochre (TAA) as an aminoacid (e.g., serine). Expression plasmids encoding a gene of interestwith one of these stop codons will express a native protein under normalconditions (see FIG. 50). If the appropriate tRNA suppressor issupplied, that stop codon will be translated (e.g., as serine whentRNA^(ser) is used) and translation will continue through any downstreamreading frame, creating a fusion protein consisting of the protein ofinterest with a specific C-terminal epitope tag (see FIG. 50). “Gene ofinterest” as used herein, refers to, for example, a nucleic acidsequence encoding a polypeptide, a protein, or an untranslated RNA,e.g., tRNA, all of which are encompassed by the term.

[0969] One non-limiting example of this stop suppression technology,termed Tag-On-Demand™ available from Invitrogen Corporation, Carlsbad,Calif., which allows expression of tagged or untagged proteins using asingle gene expression vector. In this embodiment, recombinantadenovirus vectors carrying the amber (TAG) stop suppressor tRNA genehave been developed as well as optimized protocols for use intransiently tagging a protein of interest in mammalian cells. Thespecific embodiment described here is purified, titered recombinantadenovirus (Adeno-tRNA^(TAG)) and one new GATEWAY™ Destination vector(pcDNA6.2/GFP-DEST). Tag-On-Demand™ may be used with any gene ofinterest provided the stop codon is TAG. For example, additionalInvitrogen mammalian expression vectors that are compatible withTag-On-Demand™ are listed below.

[0970] The use of the pcDNA6.2/V5 and pcDNA6.2/GFP Destination vectorsis recommended for use in Tag-On-Demand™ primarily due to thesuperiority of blasticidin as a selectable marker and the absence of theBGH polyA. In addition to the recommended vectors listed above, thefollowing three Destination vectors have also been successfully used inTag-On-Demand™ pcDNA3.2/V5-DEST, pcDNA-DEST40, and pcDNA-DEST47. Thefollowing Invitrogen vectors are all compatible with Tag-On-Demand™ andcontain a non-TAG stop codon downstream of the C-terminal epitope tag,provided the gene of interest is cloned with TAG stop in frame with theC-terminal tag: pcDNA/V5His vector family; pEF/V5His vector family;pUbC/V5His vectors; pcDNA/mycHis vector family; pEF/mycHis vectorfamily; pcDNA3.1/CT-GFP vectors; pcDNA4/TO/mycHis vectors; pGene/V5Hisvectors; pIND/V5His vectors; pcDNA5/FRT/V5His; and pEF5/FRT vectors.

[0971] Materials and Methods

[0972] Vector construction. (a) pUC12-tRNA^(TAG): Three suppressor tRNAvectors were received from Dr. Uttam RajBhandary of MassachusettsInstitute of Technology. Each suppressor tRNA vector, designated pUCtSSu+ amber, opal, and ochre, is identical except for the stop anticodon(Capone et. al. 1985, EMBO, 4(1):213-221). For convenience, the pUCtSSu+ amber vector is now referred to as pUC12-tRNA^(TAG). To create atetracycline-regulated version, referred to herein aspUC12-TO-tRNA^(TAG), two tetracycline operators (tetO₂) were cloned intothe SnaBI site in pUC 12-tRNA^(TAG) using the following annealedoligonucleotides:

[0973] tetO₂ Forward primer

[0974] 5′ GACTCGAGTCTCCCTATCAGTGATAGAGATCTCGAGGTC 3′ and

[0975] tetO₂ Reverse primer

[0976] 5′ GACCTCGAGATCTCTATCACTGATAGGGAGACTCGAGTC3′.

[0977] In italics is a unique BglII site that was introduced with theoligonucleotide. The underlined sequences are XhoI sites. All tRNAconstructs were sequence verified.

[0978] (b) pcDNA6.2/GFP-DEST: pcDNA6.2/V5-DEST was digested with ApaIand PmeI to remove the V5 tag. pcDNA3.1/lacZ-stop^(TAG)-GFP was alsodigested with ApaI and PmeI to isolate the GFP fragment. The GFP fusiontag was ligated to the pcDNA6.2 DEST vector (Invitrogen Corporation,Carlsbad, Calif. catalog # 12489-027) and transformed into DB3.1 cells.Colonies were grown on LB-Amp plates. A clone was selected that resultedin correct band fragments when digested with NdeI and then sequenceconfirmed.

[0979] (c) pENTR CAT^(TAA,TAG,TGA) The GATEWAY™ CAT entry clones werePCR amplified followed by TOPO cloning (Invitrogen Corporation,Carlsbad, Calif. product manual #25-0434) into pENTR dT. Informrationfor both vectors may be obtained by contacting Invitrogen Corporation,Carlsbad, Calif. The primer sequences used were Forward primer:5′ CACCATGGAGAAAAAAATCACTGG 3′ Reverse primer: 5′ CTGCTACGCCCCGCCCTGC3′.

[0980] The underlined sequence varied depending on which stop codon wasrequired. Plasmid constructs were sequence verified.

[0981] (d) pcDNA3.2/V5-GW/CAT^(TAA, TAG, TGA): pcDNA3.2/V5-DEST andpENTR CAT with each of the stops was recombined using LR clonase togenerate the plasmids pcDNA3.2/V5-GW/CAT^(TAA, TAG, TGA). Clones wereidentified as correct by restriction enzyme digests and sequenceconfirmed.

[0982] (e) pcDNA6.2/GFP-GW/CAT^(TAA, TAG, TGA): pcDNA6.2/GFP-DEST andpENTR CAT with each of the stops was recombined using LR clonase togenerate the plasmids pcDNA6.2/GFP-GW/CAT^(TAA, TAG, TGA). Clones wereidentified as correct by restriction enzyme digests and sequenceconfirmed.

[0983] (f) pENTR p48^(TAG): This GATEWAY™ Entry clone was obtained fromthe Ultimate™ ORFeome Collection (Invitrogen Corporation, Carlsbad,Calif.) and is referred to by several names: HS8-E6 (internal Invitrogendesignation), BC000141 (GenBank Accession number), or ORF 12 (used forconvenience). This ORF is referred to as p48 and is a human c-mycvariant (see Results section). Information for this clone may beobtained by contacting Invitrogen Corporation, Carlsbad, Calif. orGenBank.

[0984] (g) pcDNA6.2/GFP-GW/p48^(TAG): pcDNA6.2/GFP-DEST and pENTRp48^(TAG) were recombined with LR clonase to generatepcDNA6.2/GFP-GW/p48^(TAG). The recombination reaction was transformedinto TOP10 cells (Invitrogen Corporation, Carlsbad, Calif., catalog#C4040-10) and plated on LB Ampicillin plates. Colonies were picked andclones were identified as correct by restriction enzyme digests andfunctional suppression.

[0985] (h) pcDNA6.2/V5-GW/p48^(TAG): pcDNA6.2/V5-DEST and pENTRp48^(TAG) were recombined with LR clonase to generate the plasmidpcDNA6.2/V5-GW/p48^(TAG). The recombination reaction was transformedinto TOP10 cells and plated on LB Ampicillin plates. Colonies werepicked and clones were identified as correct by restriction enzymedigests and functional suppression.

[0986] (i) pENTR-TO-tRNA^(TAG): pENTR1A (Invitrogen Corporation,Carlsbad, Calif.) and pUC12-TO-tRNA^(TAG) (described in (a) above) weredigested with SalI and EcoRI. Following digests, the appropriate bandswere gel purified and ligated. Ligations were transformed into TOP10cells and plated on LB-Kanamycin plates. Clone 1 was selected followingSalI and EcoRI diagnostic digests.

[0987] (j) pENTR-tRNA8^(TAG): Primers were created to PCR amplify thetRNA gene from pUC12 TO tRNA^(TAG) with EcoRI and XbaI sequences at the5′ end, and SpeI and HindIII at the 3′ end. The primer sequences were:

[0988] Forward primer:

[0989] 5° CACCGAATTCTCTAGAGATGTCTGTGAAAAGAAACAT 3′ and

[0990] Reverse primer:

[0991] 5′ ATATAAGCTTACTAGTCCGGATTTCCTCTACCCGAGA 3′.

[0992] The tRNA PCR product was gel purified, TOPO cloned into pENTR dT,and transformed into TOP10 cells. Colonies were selected on LB Kanamycinplates. Upon confirmation of proper insertion, two separate digests wereconducted. The first digest with EcoRI and XbaI opened thepENTR-tRNA^(TAG). The second digest with EcoRI and SpeI excised the tRNAgene. Correct fragments were gel purified, the two fragments wereligated, as XbaI and SpeI have complimentary ends, thus creating a dimerof tRNA. With confirmation of proper insertion, the same two previousdigests were repeated with the dimer plasmid, fragments gel purified,ligations performed creating a tetramer. A final two digests, aspreviously described, were repeated on the tetramer, fragments gelpurified, ligations performed creating an octamer tRNA in the pENTRbackbone. (Buvoli et al., Mol. Cell. Biol. 20:3116-3124 (2000),Suppression of Nonsense Mutations in Cell Culture and Mice byMultimerized Suppressor tRNA Genes).

[0993] Adenovirus tRNA

[0994] Adenovirus carrying the suppressor tRNA^(TAG) was created using aGATEWAY™ LxR reaction. pAd/PL-DEST vector (Table 10, FIG. 9) wasrecombined with either pENTR-tRNA^(TAG) or pENTR-tRNA8^(TAG) to createpAd-tRNA^(TAG) (Table 8) or pAd-tRNA8^(TAG) expression vectors,respectively. These vectors were subsequently cut with PacI andtransfected into TREx 293 (Invitrogen Corporation, Carlsbad, Calif.,catalog #R710-07) cells to produce the initial stocks of recombinantadenovirus. Subsequent virus amplification and titering was performed in293A cells as previously described in Example 4.

[0995] Adenovirus Production and Purification

[0996] Ten T-175 flasks of 293A cells were plated in 25 ml of completemedium per flask (DMEM/10% FBS/L-Glutamine/non-essential aminoacids/penicillin/streptomycin). On the day of infection, the cells were80-90% confluent. The old media was removed and replaced with 25 ml ofcomplete media containing sufficient virus for an MOI of 5 viruses percell. Cultures were incubated overnight at 37° C. The next day, themedia was replaced with 25 ml fresh media and the cells were incubatedfor 2-3 days until >80% cytopathic effect (CPE) was observed. CPE isobvious: the cells swell, round-up, and begin to detach from the plate.At this point, the cells were gently dislodged using a 50 ml pipette andpooled in 250 ml sterile conical bottles. Cultures were centrifuged at1000 rpm for 10 minutes. The supernatant was discarded, the cell pelletwas dissolved in 5 ml of PBS and transferred to a 15 ml polypropylenetube. Cells were lysed to release virus by three freeze/thaws (−80° C.to 37° C.). Care was taken not to leave sample at 37° C. any longer thannecessary to melt it, as virus degradation is accelerated at 37° C.After the freeze/thaw cycles, 150 μl was removed for the wild type assay(see below). The lysates were then treated with DOC (deoxycholate,sodium salt, Sigma-Aldrich, St. Louis, Mo. catalogue #D 6750) toincrease the virus yields. A stock of 10% DOC was prepared in water(heat was required to get it all into solution) and DOC was added to theadenovirus lysate to a final concentration of 0.2%. The lysates wereincubated at room temperature for 30 minutes on a rotating platform.Insoluble materials were eliminated by centrifugation (3000 rpm for 15minutes in table top centrifuge), and the crude high titer viralsupernatant (CHT) was transferred to a fresh tube. MgCl₂ was added to 5mM final and virus was stored at −80° C. or cesium chloride purified(see below).

[0997] Cesium chloride step-gradient ultracentrifugation purifies andconcentrates the recombinant adenovirus by eliminating cellularcontaminants in order to achieve optimum efficiency and minimal toxicityof adenovirus gene delivery. Two cesium chloride (Molecular Biologygrade CsCl, Sigma-Aldrich, St. Louis, Mo., catalog #C3032) solutionswere prepared with the following densities: 1.4 g/ml and 1.25 g/ml in 10mM Tris (pH 8.0). [1.4 g/ml=38.83 g CsCl+61 ml 10 mM Tris and 1.25g/ml=26.99 g CsCl+73 ml 10 mM Tris] These weight/volume densities wereverified by weighing 1 ml of each solution. Density was adjusted byadding more cesium chloride or 10 mM Tris as needed to achieve correctdensity. Each solution was filter sterilized and stored at roomtemperature.

[0998] To prepare the step-gradient, 2.5 ml of 1.25 g/ml CsCl solutionwas placed in one ultracentrifuge tube (SW41 Beckman centrifuge tubes,thin wall polyallomer 14×89, part#331372) and then carefully underlayedwith 2.5 ml of 1.4 g/ml CsCl solution. A long glass Pasteur pipette wasused for underlaying. Next, the step-gradient was gently overlaid withthe 5 ml of crude high titer viral lysate and carefully move intoBeckman SW41 centrifuge rotor. Samples were spun at 35,000 rpm for onehour and ten minutes at 20° C. After ultracentrifugation, cellularlipids and cytoplasmic debris remained at the top of the tube, and thecloudy adenovirus band migrated near the interface of the two CsCllayers. The virus band was very obvious to the naked eye. The tube wasclamped to a ring stand and the sides of the tube were wiped with 70%ethanol. The virus band was harvested using a 3 ml syringe fitted with a20 or 21 gauge needle by side puncture. Virus was transferred to a 15 mlconical tube and the volume estimated. Glycerol was added to 10% final.

[0999] The cesium chloride in the recombinant adenoviral preparation wasremoved by four rounds of dialysis. For each ultracentrifuge tube fourliters of dialysis buffer was required. The dialysis buffer consisted of10 mM Tris (pH 7.5), 1 mM MgCl₂, 150 mM NaCl, and 10% glycerol final andwas kept at 4° C. Buffer was prepared the day before dialysis and placedin the cold room with a stir bar overnight. The recovered viral band wasdialyzed at 4° C. using. a Spectrum Specta/Por CE Float-A-Lyzer with amolecular weight cut-off (MWCO) of 300,000 Dalton (Fischer 3 ml size#08-700-51 or 5 ml size #08-700-64). The virus preparation was dialyzedfour times. Each dialysis was conducted for one hour and in one liter,with constant gentle stirring. The final virus product was removed fromFloat-A-Lyzer with the plastic pipette provided and aliquotted intoeppendorf tubes. Aliquots of virus were stored at −80° C. and multiplefreeze/thaws were avoided.

[1000] Typical titers from a 10-flask cesium chloride preparation rangefrom 7×10⁹ to 6.5×10¹⁰ pfu/ml. The volume of purified recombinantadenovirus obtained is typically around 1.0 ml, making the total virusyield from 10-flasks to be 7×10⁹ to 6.5×10¹⁰ pfu. This stock containsenough material for one 96-well plate using an MOI of 50.

[1001] Wild-Type Assay

[1002] The “supernatant rescue assay” is performed to detect wild-typeadenovirus contamination in recombinant adenovirus stocks using standardprocedures (Dion et al., J. Virol. Methods 56:99-107 (1996)).

[1003] Reporter Cell Line

[1004] pcDNA6/FRT/V5 was digested with PstI and PmeI to remove thenucleic acid sequence encoding the V5 tag. pcDNA3.1 lacZ stop^(TAG) GFPwas digested with PstI and Pme I to isolate the fragment containing lacZstop^(TAG) GFP. The above fragments were gel purified, ligated, andtransformed into TOP10 cells. The resulting reporter plasmid,pcDNA6/FRT/lacZ-stop^(TAG)-GFP, was verified by diagnostic digests andsequencing. FlpIn CHO cells (Invitrogen Corporation, Carlsbad, Calif.,catalog #R758-07) were co-transfected with the vector pcDNA6/FRT/lacZstop^(TAG) GFP and pOG44 (invitrogen Corporation, Carlsbad, Calif.,catalog #V6005-20) at a ratio of 1:10. Blasticidin selection was started4 days post transfection at a concentration of 15 μg/ml. Selection wascomplete after 24 days.

[1005] Co-transfections

[1006] Six-well plates were seeded with cells one day prior totransfections. Cells tolerate transfections best if seeded at a densitythat allowed for greater than 90% confluency the day of co-transfection.Co-transfections were conducted for a minimum of 5 hours and up toovernight. The lipid complexes were then removed and replaced with freshmedia. Co-transfections were optimized using 1.5 μg suppressor tRNAplasmid and 0.5 μg of the corresponding reporter vector combined in 250μl of Opti-MEM® I Reduced Serum Medium (OPTI-MEM) at room temperaturefor 5-10 minutes. Six microliters of Lipofectamine™ 2000 was combinedwith 250 μl of OPTI-MEM and allowed to sit at room temperature for 5minutes before combining with the DNA mixture. The DNA-lipid complex wasallowed to form for 20 minutes at room temperature. Subsequently, theDNA-lipid complex was added to the cells in wells containing 2 ml ofmedia. Suppression in a GFP fused expression vector could be observedthe following day and up to 72 hours post transfection. Cells weretypically lysed and harvested twenty-four hours post transfection withIGE PAL CA630 lysis buffer (Sigma-Aldrich, St. Louis, Mo., catalog#I-3021) or RIPA lysis buffer (10 mM Tris (8.0), 150 mM NaCl, 0.1% SDS,1.0% NP-40 (or Triton X-100), 1.0% deoxycholate, 2 mM EDTA) withleupeptin, pepstatin, and PMSF.

[1007] Transductions

[1008] Cells were transduced with suppressor tRNA for a minimum of fivehours to a maximum of overnight in a total of 1 ml media in a six wellformat. Upon completion of transduction the virus was removed and 2 mlof fresh media was added. The cells were then transfected overnight orthe following day. Cells were typically lysed and harvested three dayspost transduction with IGE PAL CA630 lysis buffer or RIPA lysis bufferwith leupeptin, pepstatin, and PMSF.

[1009] Westerns

[1010] Cell lysates were centrifuged at maximum speed for 1-2 minutes.Lysates were then transferred to new tubes and pellet discarded. ABradford protein assay was conducted to determine the proteinconcentration of each lysate. For western blotting, 10-30 μg of proteinwas loaded on a gel. Determination of percent suppression was performedusing 6% Tris Glycine gels for western blotting of β-galactosidasefusion proteins to maximize resolution of high molecular weightproteins. For CAT, GFP and V5 blots, 4-20% Tris Glycine gels were used.Proteins from gels were transferred to 0.45 μm nitrocellulose usingwestern blotting technique. Various antibodies were used in detection ofproteins: anti-βgal at 1:5000, anti-CAT at 1:5000, anti-GFP at 1:5000,anti-V5 at 1:5000. Western Breeze kits and antibodies from InvitrogenCorporation, Carlsbad, Calif. were used throughout.

[1011] QC Assay for Manufactured Virus

[1012] Virus produced in manufacturing should be screened for wild typevirus, cesium chloride purified and plaque-assay titered. For theactivity assay, COS-7 cells (ATCC #CRL-1651) were seeded at a density of3×10⁵ cells in a 6 well format with 2 ml of DMEM containing 10% FBS, 1%L-glutamine, and 1% Pen/Strep. The following day, the media wasaspirated and 1 ml was added back to the culture wells to be transduced.CsCl purified Ad-tRNA8^(TAG) was added to each well at an MOI of 0, 25,50 and 100. The transductions were allowed to proceed for 5-6 hours at37° C. Following the transduction period, the media containing the viruswas removed and 2 ml of fresh media was added back to each well. Thetransduced cells were allowed a day to recover before transfection ofreporter plasmid. For each transfected well, two micrograms ofpcDNA6.2/GFP-GW/p48^(TAG) (or pcDNA3.1/lacZ-STOP^(TAG)-GFP) expressionplasmid was diluted in 250 μl of OPTI-MEM and incubated at roomtemperature for 5 minutes. 6 μl of Lipofectamine™ 2000 was diluted in250 μl of OPTI-MEM and incubated at room temperature for 5 minutes. TheDNA and lipid dilutions may also be set-up in batch for the four wellsto be transfected. The DNA and lipid were then combined and incubated atroom temperature for 20 minutes to complex before adding to COS-7 cellspreviously transduced with Ad-tRNA8^(TAG). The DNA-lipid complexremained on the cells between 5 to 18 hours before being removed andfresh media added to the cells. GFP fluorescence was observed on days1-3 post transduction. The cells were lysed and harvested with ice cold150-200 μl of RIPA lysis buffer (10 mM Tris (8.0), 150 mM NaCl, 0.1%SDS, 1.0% NP-40 (or Triton X-100), 1.0% deoxycholate, 2 mM EDTA)containing leupeptin, pepstatin, and PMSF on day 3 post transduction.The lysates were then centrifuged for 5 minutes at maximum speed(preferably at 4° C.). Lysates were transferred to new 1.5 ml eppendorftubes and frozen at −80° C. if not used immediately for westernblotting. Following western blotting for anti-myc (oranti-β-galactosidase, depending on the expression plasmid used)densitometry was performed on the Fujifilm LAS-1000 Densitometer usingthe software Image Reader LAS-1000 Lite v1.0 and imageGauge v.254.Percent suppression was calculated by dividing the density of the upperband by the total (lower plus upper band). For the purpose of thisexample, 50% suppression was the desired level of suppression.

[1013] Results and Discussion

[1014] All three possible human tRNA suppressors (TAG, TAA and TGA) werecreated by mutating the anticodon of the human tRNA serine gene (Caponeet al., EMBO, 4:213-221 (1985)). This work was performed in thelaboratory of Dr. RajBhandary, who also provided the pUC12-based vectorscontaining each of the three tRNA^(ser) suppressors. Bacterial tRNAsuppressors had been identified many years previously, but the use of amammalian tRNA suppressor allows stop suppression in mammalian cellswithout the need to co-express the cognate tRNA charging enzyme.

[1015] The efficiency of each tRNA suppressor was tested in severalco-transfection experiments (FIGS. 51A-B). Three GATEWAY™ entry cloneswere created, with CAT as the gene of interest (GOI), followed by eachof the three stop codons (pENTR-CAT^(TAA), pENTR-CAT^(TAG) andPENTR-CAT^(TGA)). These entry clones were LR crossed into eitherpcDNA3.2/V5-DEST or pcDNA6.2/GFP-DEST, thus placing either V5 or GFPdownstream (and in frame) of the native CAT ORF. These Destinationvectors also have all three stop codons, in frame, downstream of theC-terminal tag. Having all three stop codons assures termination oftranslation after the tag, regardless of which tRNA suppressor is used.

[1016] V5 Epitope Tag-On-Demand™

[1017] CHO cells were co-transfected with one of these expressionvectors: pcDNA3.2/V5-GW/CAT^(TAA), -GW/CAT^(TAG) or -GW/CAT^(TGA) in thepresence or absence of its cognate tRNA suppressor: pUC12-tRNA^(TAA),pUC12-tRNA^(TAG) or pUC12-tRNA^(TGA) (FIG. 51A). Western blot analysisusing antibodies against the V-5 epitope revealed easily detectableV5-epitope-tagged protein in the presence of tRNA suppressor (leftpanel), which was further illustrated by the “shift” up of CAT proteinon the anti-CAT western blot (right panel). The efficiency ofsuppression can be calculated by using densitometry to scan theintensity of the shifted and un-shifted bands. In this experiment aswell as others not described herein, the TAG stop suppressor was clearlysuperior to the other two, demonstrating a >70% conversion of native CATto CAT-V5 in the presence of the suppressor (FIG. 51A, right panel,anti-CAT blot), as compared to only 44% and 53% for TAA and TGA,respectively.

[1018] GFP Tag-On-Demand™

[1019] 293FT cells (Invitrogen Corporation, Carlsbad, Calif., catalog#R700-07) were co transfected with one of the three expression vectors(pcDNA6.2/GFP-GW/CAT^(TAA), -GW/CAT^(TAG) or -GW/CAT^(TGA)) and one ofthe pUC12-tRNA vectors (FIG. 51B). Anti-CAT western blotting showed aclear shift of native CAT up to CAT-GFP when the correct tRNA wassupplied. Again, tRNA^(TAG) demonstrated superior stop suppressioncompared to the other two tRNA suppressors. It is also important to notethat the stop suppression and protein tagging is very specific. In otherwords, when the incorrect tRNA suppressor is supplied, no stopsuppression is observed and only native protein is expressed (forexample, see TAA CAT reporter with tRNA^(TAG) or tRNA^(TGA), FIG. 51B).The specificity of the suppression is further demonstrated with adifferent reporter vector, pcDNA3.1/lacZ-stop^(TAG)-GFP (FIG. 52). Onlyin the presence of the correct tRNA suppressor (pUC12-tRNA^(TAG)) wasthe β-galactosidase-GFP fusion protein expressed resulting in detectableglowing in the cells (center panels, FIG. 52). When either of the othersuppressors is used (tRNA^(TAA) or tRNA^(TGA)), no suppression of theTAG stop occurs, no β-galactosidase-GFP is expressed and no glowing isobserved in the transfected cells (left and right panels FIG. 52).

[1020] Adenovirus-tRNA Delivery

[1021] Since Tag-On-Demand™ is primarily designed for the transienttagging of proteins, an ideal delivery method of the suppressor gene tomammalian cells is using a recombinant adenovirus. Adenovirus has a verybroad tropism for different mammalian cell types and transductionefficiencies can approach 100% (for review see Russell 2000, Update onadenovirus and its vectors. J. Gen. Virol., 81:2573-2604). Furthermore,since the virus does not stably integrate into the host genome,expression is transient. In actively dividing cells (24 hour doublingtime), gene expression from adenoviral vectors is typically detectedwithin 24 hours and persists for 7-8 days.

[1022] The tRNA^(TAG) gene was cloned into pENTR to createpENTR-tRNA^(TAG), and this was used in a GATEWAY™ LR reaction withpAd/PL-DEST (Table 10, FIG. 9) to create pAd-tRNA^(TAG). Severallarge-scale preparations of virus were performed and functional testingwas done. Adenovirus proved to be a very efficient way of delivering thetRNA, however preliminary experiments required MOIs (multiplicity ofinfection) of several hundred to deliver biologically relevant amountsof the tRNA. The goal was to achieve at least 50% suppression using anMOI of 50 in COS cells transfected with one of the reporter genes. It isbelieved that the tRNAs must compete with endogenous protein “stopfactors” occupying the stop codon, which may explain the more efficientsuppression in the presence of multiple copies of the nucleic acidmolecule encoding the suppressor tRNA sequence. In an attempt to reducethe number of viral particles required for efficient suppression, eightcopies of the tRNA gene were cloned into pENTR (calledpENTR-tRNA8^(TAG)) and recombined into the adenovirus promoterlessDestination vector. This new adenovirus (Adeno-tRNA8^(TAG)) was comparedwith the original monomer virus (Adeno-tRNA^(TAG)) for stop suppression(FIG. 53). As shown by both fluorescent microscopy (upper panels) andanti-β-galactosidase western blotting (lower panel), a modest increasein suppression efficiency was observed with the 8-mer tRNA, and thesesuppression levels are as good as those seen with the plasmid-based tRNA(lanes 2 and 4). Indeed, in all subsequent experiments, theAd-tRNA8^(TAG) transduction performed as well or better than apUC-tRNA^(TAG) plasmid transfection making this recombinant adenovirusconfiguration particularly suitable for the methods of this invention.

[1023] The initial adenovirus experiments used crude adenoviruspreparations that still contained all of the debris from the lysedproducer cells (roughly 10⁸ cells lysed in 5 ml PBS). This material wasfunctional but resulted in unacceptably high toxicity to the targetcells. A variety of purification methods were evaluated to attempt toremove the toxic components from the active adenovirus. Large poredialysis (300,000 MWCO), sucrose density gradient purification, and HPLCwere evaluated for use in the methods of this invention, as weretraditional cesium chloride purification and two commercially availableadenovirus purification columns (ViraPur, Carlsbad, Calif. and PureSyn,Malvern, Pa.). It was deduced that a modified, single-round cesiumchloride step gradient purification (described above) was the leastexpensive option that gave the highest yields of active virus andexhibited the lowest toxicity on the target cells, making this methodparticularly suitable for use in the methods of this invention.

[1024] Ultimate™ ORF Collection Tag-On-Demand™

[1025] The invention described herein is compatible for use with anygene or ORF of interest providing the stop codon is recognized by theprovided suppressor tRNA. This stop codon may be native to the gene ofinterest, or it may be inserted by standard molecular biologytechniques, such as described herein. Particularly suited for use in themethods of this invention are clones in the Ultimate™ ORF collectionavailable from Invitrogen Corporation, Carlsbad, Calif. This collectionof genes is provided as GATEWAY™ Entry clones containing the native ORFwith a TAG stop codon. Tag-On-Demand™ will allow quick and easydetection of expressed protein products (either via V5 or GFP tagging)without needing to generate antibodies against the native protein orrecloning the gene to a separate expression vector.

[1026] To demonstrate the usefulness of Tag-On-Demand™ with ORFs fromthe Ultimate™ ORF collection, three human ORFs were chosen andrecombined into either pcDNA6.2/GFP-DEST or pcDNA6.2/V5-DEST.Transduction of cells with the Ad-tRNA8^(TAG) followed by transfectionof the cells with the ORF-GFP expression clone resulted in easilyvisible GFP positive cells (FIG. 54, upper panels). Significantly, theproteins retained their normal subcellular localization that was easilydetectable using fluorescence microscopy. ORF6 (BC003357) codes for aCGI-130-like protein (23.4 kD) that is primarily cytoplasmic, withnuclear exclusion observed in some cells. ORF7 (BC000997) codes for ahuman mRNA splicing factor (27.4 kD) and was clearly localized to thenucleus, as expected. ORF12 (BC000141) codes for a truncated form ofc-myc (48.8 kD) that is also nuclear, with specific targeting topunctate nucleolar structures. ORF12 is referred to as p48^(TAG) above,and can be the positive expression control for use in kits, as providedin the methods of the present invention. In this example, thisexperiment was performed by first transducing the cells with adenovirusfor 6 hours, followed by transfecting with the reporter plasmidovernight. Alternatively, transduction and transfection may be performedtogether, or, transfection first followed by transduction. All threemethods resulted in good suppression, though transduction followed bytransfection to may achieve the best suppression and the least toxicity.

[1027] V5 epitope tagging of the ORFs was also successful using themethods of the present invention. Each expressed protein was easilydetectable via anti-V5 western blotting, in the presence of thetRNA^(TAG), and migrated at the correct molecular weight (FIG. 54, lowerpanel). The addition of the V5 epitope adds approximately 4.2 kD to theprotein of interest. ORF-V5 expression levels were comparable tolacZ-stop^(TAG)-V5 suppressed with tRNA^(TAG), and surprisingly as goodas a true V5 fusion protein, GFP-V5, expressed constitutively frompcDNA/GFP-V5 (last lane). This experiment was performed byco-transfection of the ORF-V5 plasmid with pUC12-tRNA^(TAG), howeversimilar results are obtained using Ad-tRNA8^(TAG).

[1028] It is an important aspect of the invention that ORF expressionvectors such as these may express the native protein under normalconditions, allowing the study of its native function. Application ofTag-On-Demand™ allows the use of the exact same expression construct totransiently create tagged versions of the protein. This aspect may beuseful for verification of protein expression, analysis of itssubcellular localization and even FACSorting of expressing cells withouthaving to generate antibodies to the specific protein or re-cloning theORF as a true C-terminal fusion.

[1029] Tag-On-Demand™ can be used on Both Transient and Stable GeneTargets

[1030] One aspect of the present invention is the transient expressionof the protein of interest with a tag to verify expression orlocalization, as described herein. Another aspect of the presentinvention is to stably express a protein of interest, as demonstrated bythe following experiment. Flp-In CHO cells stably expressing a singlecopy of pcDNA6/FRT/lacZ-stop^(TAG)-GFP were transduced withAdeno-tRNA8^(TAG) at various MOIs (FIG. 55A). Anti-lacZ western blottingrevealed a dose-dependent increase in stop suppression with increasingamounts of Adeno-tRNA^(TAG), and clearly demonstrates thatTag-On-Demand™ can be used to C-terminally tag stably expressed genes.This experiment shows that C-terminally-tagged recombinant protein isproduced at all MOIs tested. As the cells are transduced with increasingMOI, the % suppression increases; however, the amount of totalrecombinant protein produced (untagged and GFP-tagged protein) remainsnearly equivalent. The band labeled with an asterisk results from theendogenous lacZ-Zeocin™ fusion present in Flp-In™-CHO cells and isderived from the construct used to create the Flp-In™-CHO cell line.In aparallel experiment, COS cells were transduced with the same range ofMOIs for 6 hours, followed by transient transfection of thelacZ-stop^(TAG)-GFP expression vector (FIG. 55B). A dose-dependentincrease in suppression with increasing amount of virus was shown, andan MOI as low as 19 can give suppression levels greater than 50%,demonstrating the efficiency of the invention. This experiment showsthat at all MOIs tested, the % suppression achieved is >60%, resultingin production of significant levels of GFP-tagged recombinant protein.As the cells are transduced with increasing MOI, the % suppressionincreases; however, the amount of total recombinant protein produced(untagged and GFP-tagged protein) decreases. This may be indicative ofcellular toxicity as a result of the addition of increasing amounts ofvirus.

[1031] Tag-On-Demand™ in Common Mammalian Cell Types

[1032] Five commonly used mammalian cells were chosen to evaluateefficiency of Tag-On-Demand™: BHK, CHO, COS, HeLa and HT1080. Cells weretransduced with Adeno-tRNA^(TAG) at an MOI of 50 for 6 hours, followedby transient transfection with the lacZ-stop^(TAG)-GFP expressionvector. In all cell types tested, Tag-On-Demand™ clearly producedsufficient lacZ-GFP fusion protein to easily detect GFP fluorescence ineach cell type (FIG. 56). The slight toxicity observed in theseexperiments most likely arose from residual cesium chloride in thepurified adenovirus preparation.

[1033] Summary

[1034] One embodiment of the present invention is exemplified by theTag-On-Demand™ system. Tag-On-Demand™ is a system that uses recombinantadenovirus to deliver a tRNA suppressor gene that results in detectionof proteins from the Invitrogen Corporation, Carlsbad, Calif. Ultimate™ORF collection. As described in the results section, Tag-On-Demand™ isprimarily designed for: a) transient detection and localization ofexpressed protein products, and b) sorting or analysis of expressingcells. Any of the three stop codons normally utilized by cells can besuppressed with the correct tRNA, with TAG stop suppression being mostefficient. Adenovirus has been chosen as the tRNA delivery method andwas shown to be as good or better than plasmid delivery. Adenovirus isan ideal method for delivering the tRNA genes for a number of reasons:

[1035] Adenovirus has broad mammalian cell tropism. Adenovirus binds tothe CAR receptor present on most mammalian cells. It is important tonote, however, that not all cell types express equal levels of therequired CAR receptor, so efficiency may vary from one cell type toanother. Fortunately, suppression efficiency can be increased byapplying more virus (see FIGS. 55A-B). Like any E1 deleted recombinantadenoviral vectors, use of the Tag-On-Demand™ adenovirus in 293 cells orin any mammalian cell that is expressing the E1 gene of adenovirus willlead to virus replication and possible death of the target cell.

[1036] Adenovirus does not stably integrate into the target cell'sgenome and its expression is transient. Stable delivery or constitutiveexpression of the tRNA suppressor would most likely be toxic to the cellsince one third of the endogenous stop codons would be suppressed,resulting in the addition of extra amino acids to the C-termini of manycellular proteins.

[1037] Adenovirus is a very stable virus and can be produced in largequantities. Manufacturing of the virus may include a single round ofcesium chloride purification (see Materials and Methods), which yields apure viral stock with minimal toxicity to the target cell.

[1038] In summary, the Tag-On-Demand™ system allows a single expressionvector to express either native protein or C-terminally tagged protein.The system is completely compatible with the GATEWAY™ cloning technologyand Ultimate™ ORF collection. The present invention is particularlysuited for use with the pcDNA6.2/V5 and pcDNA6.2/GFP Destinationvectors, however a person of skill in the art would readily recognizethat a variety of other Invitrogen vectors as well as others are alsocompatible with Tag-On-Demand™, including many TOPO vectors, myc and6×-His vectors, T-REx and Flp-In as described in the Materials andMethods. Further provided in the present invention is the ability toclone any downstream “tag” for fusing to any protein of interest,provided there is a non-TAG stop codon at the end of the C-terminal tag.

Example 15 Tag-on-Demand™ GATEWAY® Vectors

[1039] In some embodiments, the present invention provides nucleic acidmolecules (e.g., vectors) that may be used to express fusionpolypeptides (e.g., polypeptides comprising a sequence of interest andat least one additional polypeptide sequence). Non-limiting examples ofvectors suitable for use in the present invention are GATEWAY®-adapteddestination vectors. Such vectors may be used for high-level expressionof native and C-terminally-tagged polypeptides from the same nucleicacid molecules. In some embodiments, such vectors may be used to expresspolypeptides (which may be fusion polypeptides) in mammalian cells. Suchvectors may be used to express fusion polypeptides by introducing thevectors into a host cell and also introducing into the host cell asource of a suppressor tRNA. Any suitable source of a suppressor tRNAmay be used (e.g., plasmids, linear nucleic acid molecules, viruses,etc.) In some embodiments, the present invention provides an adenovirusthat expresses one or more suppressor tRNA molecules and/or one or morecopies of a suppressor tRNA molecule. Methods that employ an adenovirusexpressing tRNAs may be referred to herein as the Tag-on-Demand™ System.

[1040] One or more of the following commercially available items may beused in connection with the methods of the invention. These items arelisted with their Invitrogen Corporation, Carlsbad, Calif. catalognumber in parenthesis: Tag-On-Demand™ Suppressor Supernatant (K400-01 orK405-01); Gateway® LR Clonase™ Enzyme Mix (11791-019 or 11791-043);Library Efficiency® DB3.1™ Competent Cells (11782-018); One Shot® TOP10Chemically Competent E. coli (C4040-03); Library Efficiency DH5α™Chemically Competent E. coli (18263-012); Blasticidin (R210-01);Ampicillin (Q100-16); S.N.A.P.™ MidiPrep Kit (K1910-01); Lipofectamine™2000 (11668-027 or 11668-019); and Phosphate-Buffered Saline (PBS), pH7.4 (10010-023).

[1041] In some embodiments, the present invention provides methods ofproducing fusion polypeptides comprising a polypeptide sequence ofinterest fused to one or more additional polypeptide sequences. If afusion polypeptide is produced (e.g., from pcDNA™6.2/V5-DEST orpcDNA™6.2/GFP-DEST), the fusion polypeptide can be detected using anantibody that binds to one or more of the additional polypeptidesequences (e.g., to the V5 epitope or to GFP). Commercially availableantibody preparations can be used, for example, those available fromInvitrogen Corporation, Carlsbad, Calif. such as Anti-V5 Antibody(catalog # R960-25), Anti-V5-HRP Antibody (catalog # R961-25),Anti-V5-AP Antibody (catalog # R962-25), Anti-V5-FITC Antibody (catalog# R963-25), GFP Antiserum (catalog # R970-01).

[1042] In some embodiments, methods of the invention may be used toexpress a fusion polypeptide comprising all or a portion of p64. Todetect the p64 (human c-myc) protein expressed using methods andmaterials of the invention, commercially available Anti-myc Antibodiesmay be used (e.g., Invitrogen Corporation, Carlsbad, Calif. Anti-mycAntibody catalog no. R950-25, Anti-myc-HRP Antibody catalog no. R951-25,Anti-myc-AP Antibody catalog no. R952-25, and/or Anti-myc-FITC Antibodycatalog no. R953-25).

[1043] Examples of nucleic acid molecules that may be used in thepractice of the invention include, but are not limited to,pcDNA™6.2/V5-DEST (7.3 kb) and pcDNA™6.2/GFP-DEST (8.0 kb), which aredestination vectors adapted for use with GATEWAY® Technology (InvitrogenCorporation, Carlsbad, Calif.) and allow high-level, constitutiveexpression of recombinant polypeptides in mammalian cells. The vectorsare designed for use with a suppressor tRNA producing nucleic acidmolecule (e.g., Invitrogen's Tag-on-Demand™ System), which allowsexpression of both native and C-terminally-tagged recombinantpolypeptide from the same expression construct.

[1044] The pcDNA™6.2/V5-DEST and pcDNATm6.2/GFP-DEST vectors enableexpression of recombinant polypeptide containing a choice of C-terminaltags. The pcDNATm6.2/V5-DEST vector encodes the V5 epitope for detectionof recombinant polypeptide using the Anti-V5 antibodies. A plasmid mapis provided as FIG. 57 and the sequence of this vector is provided asTable 28. The pcDNA™6.2/GFP-DEST vector encodes the Cycle-3 GFP forfusion to a polypeptide sequence of interest and use as a reporter gene.A plasmid map of this vector is provided as FIG. 58 and the sequence ofthis vector is provided as Table 29.

[1045] The pcDNA™6.2/V5-DEST and pcDNA™6.2/GFP-DEST vectors contain thefollowing features: human cytomegalovirus (CMV) immediate early promoterfor high-level constitutive expression of the gene of interest in a widerange of mammalian cells (Andersson, S., et aL, J. Biol. Chem.264:8222-8229 (1989); Boshart, M., et al., Cell 41:521-530 (1985);Nelson, J. A., et al., Molec. Cell. Biol. 7:4125-4129 (1987)); tworecombination sites, attR1 and attR2, downstream of the CMV promoter forrecombinational cloning of the DNA sequence of interest from an entryclone; the chloramphenicol resistance gene (Cm^(R)) located between thetwo attR sites for counterselection; the ccdB gene located between theattR sites for negative selection; the C-terminal V5 epitope fordetection of the recombinant polypeptide of interest (inpcDNA™6.2/V5-DEST only) (Southern, J. A., et al., J. Gen. Virol.72:1551-1557 (1991)); the C-terminal cycle-3 Green Fluorescent Protein(GFP) gene for fusion of the recombinant polypeptide of interest to areporter (in pcDNA™6.2/GFP-DEST only) (Chalfie, M., et al., Science263:802-805 (1994); Crameri, A., et al., Nature Biotechnol. 14:315-319(1996)); the Herpes Simplex Virus thymidine kinase (TK) polyadenylationsequence for efficient transcription termination and polyadenylation ofmRNA (Cole, C. N., and Stacy, T. P., Mol. Cell. Biol. 5:2104-2113(1985)); the Blasticidin resistance gene for selection of stable celllines (Kimura, M., et al., Biochim. Biophys. ACTA 1219:653-659 (1994));the pUC origin for high-copy replication and maintenance of the plasmidin E. coli; the ampicillin (bla) resistance gene for selection in E.coli. In one alternative of this aspect of the invention, thechloramphenicol resistance gene in the cassette can be replaced by aspectinomycin resistance gene (see Hollingshead et al., Plasmid13(1):17-30 (1985), NCBI accession no. X02340 M10241), and the pcDNAdestination vector containing attP sites flanking the ccdB andspectinomycin resistance genes can be selected onampicillin/spectinomycin-containing media. Use of spectinomycinselection instead of chloramphenicol selection may result in an increasein the number of colonies obtained on selection plates, indicating thatuse of the spectinomycin resistance gene may lead to an increasedefficiency of cloning from that observed using cassettes containing thechloramphenicol resistance gene.

[1046] The location in the plasmid sequence of pcDNA™6.2/V5-DEST (7341nucleotides) of the features discussed above are: CMV promoter bases232-819; T7 promoter/priming site bases 863-882; attR1 site bases911-1035; ccdb gene bases 1464-1769 (c); chloramphenicol resistance genebases 2111-2770 (c); attR2 site bases 3051-3175; V5 epitope bases3201-3242; V5 reverse priming site 3210-3230; TK polyadenylation signalbases 3269-3540; fl origin 3576-4004; SV40 early promoter and origin4031-4339; EM7 promoter bases 4394-4460; Blasticidin resistance genebases 4461-4859; SV40 early polyadenylation signal bases 5017-5147; pUCorigin bases 5530-6200 (c); Ampicillin (bla) resistance gene bases6345-7205 (c); bla promoter bases 7206-7304 (c) where (c) indicatespresent on the complementary strand.

[1047] The location in the plasmid sequence of pcDNA™6.2/GFP-DEST (7995nucleotides) of the features discussed above are: CMV promoter bases232-819; T7 promoter/priming site bases 863-882; attR1 site bases911-1035; ccdB gene bases 1464-1769 (c); Chloramphenicol resistance genebases 2111-2770 (c); attR2 site bases 3051-3175; Cycle-3 GFP bases3195-3908; GFP reverse priming site 3303-3324; TK polyadenylation signalbases 3923-4194; fl origin 4230-4658; SV40 early promoter and origin4685-4993; EM7 promoter bases 5048-5114; Blasticidin resistance genebases 5115-5513; SV40 early polyadenylation signal bases 5671-5801; pUCorigin bases 6184-6854 (c); Ampicillin (bla) resistance gene bases6999-7859 (c); bla promoter bases 7860-7958 (c), where (c) indicates thefeature is present on the complementary strand.

[1048] In some embodiments, positive control nucleic acid molecules(e.g., plasmids may be used in conjunction with the methods of theinvention. A suitable positive control nucleic acid molecule is onecomprising a nucleic acid sequence encoding two polypeptide sequences inthe same reading frame and having a stop codon in between the sequences.For example, the polypeptide encoded 3′ to the stop codon may have adetectable activity (i.e., enzymatic activity, fluorescent activity,binding activity, etc.). Examples of suitable control nucleic acidmolecules include, but are not limited to, pAd/CMV/V5-GW/lacZ,pcDNA™6.2/V5-GW/p64^(TAG) and pcDNA™6.2/GFP-GW-p64^(TAG), which wereprepared from the corresponding vectors by conducting an L×R reactionwith an entry vector containing the indicated coding sequence (i.e.,lacZ or p64 coding sequence (also known as c-myc). Plasmid maps of thecontrol vectors pcDNA™6.2/V5-GW/p64^(TAG) and pcDNA™6.2/GFP-GW-p64^(TAG)are provided as FIGS. 59 and 60 respectively.

[1049] The GFP gene used in the pcDNA™6.2/GFP-DEST vector is describedin Crameri, A., et al., Nature Biotechnol. 14:315-319 (1996). In thispaper, the codon usage was optimized for expression in E. coli and threecycles of DNA shuffling were used to generate a mutant form of GFP thatexpresses well in mammalian cells and has excitation and emission maximathat are the same as wild-type GFP (395 nm and 478 nm for primary andsecondary excitation, respectively, and 507 nm for emission) anda >40-fold increase in fluorescent yield over wild-type GFP. This mutantGFP is referred to as Cycle-3 GFP to differentiate it from wild-typeGFP.

[1050] Materials and methods of the invention (e.g., The Tag-on-Demand™System, Invitrogen Corporation, Carlsbad, Calif.) facilitate transientexpression of C-terminally-tagged and untagged recombinant polypeptidesfrom a single expression construct such as one prepared using GATEWAY™.The System is based on stop suppression technology originally developedby RajBhandary and colleagues (Capone, J. P., et al., EMBO J. 4:213-221(1985)), and consists of two major components: an expression vector intowhich the gene of interest will be cloned and a nucleic acid molecule(or composition comprising such a nucleic acid molecule) expressing oneor more suppressor tRNAs (e.g., the Tag-on-Demand™ SuppressorSupernatant). The vector (e.g., pcDNA™6.2/V5-DEST or pcDNA™6.2/GFP-DEST)must be in a configuration that is compatible with expression ofC-terminally-tagged recombinant polypeptide by introducing a suppressortRNA to suppress a stop codon (e.g., by using the Tag-on-Demand™System). In one non-limiting embodiment, (i.e., the Tag-on-Demand™Suppressor Supernatant) a suppressor tRNA molecule may be introducedinto a host cell by transducing the host cell with areplication-incompetent adenovirus containing the human tRNA^(ser)suppressor. This tRNA suppressor has been mutated to recognize the TAG(amber stop) codon and decode it as a serine. When added to mammaliancells, the Tag-on-Demand™ Suppressor Supernatant is transduced andprovides a transient source of the tRNA^(ser) suppressor.

[1051] When an expression vector encoding a gene of interest with theTAG stop codon is transfected into mammalian cells, the stop codon willbe translated as serine, allowing translation to continue through anydownstream reading frame (e.g., a C-terminal tag), and resulting inproduction of a fusion polypeptide containing the polypeptide encoded bythe gene of interest fused to the amino acids encoded 3′ to the stopcodon (e.g., a marker or tag sequence). One skilled in the art willappreciate that, in similar fashion, a nucleic acid molecule (e.g., areplication-incompetent adenovirus) expressing a suppressor tRNA thatsuppresses TAA (ochre) or TGA (opal) stop codons can be prepared andused in the practice of the present invention.

[1052] To recombine a DNA sequence of interest into a nucleic acidmolecule of the invention (e.g., pcDNATm6.2/V5-DEST orpcDNA™6.2/GFP-DEST), an entry clone containing the DNA comprising asequence of interest may prepared. In an entry clone, a sequence ofinterest may be flanked by recombination sites (e.g., sites compatiblewith those in one or more destination vector). Many entry vectors areavailable from Invitrogen to facilitate generation of entry clones.Examples include, but are not limited to, pENTRID-TOPO® (catalog numberK2400-20), pENTR/SD/D-TOPO® (catalog number K2420-20), pENTR™1A (catalognumber 11813-011), pENTR™2B (catalog number 11816-014), pENTR™3C(catalog number 11817-012), pENTR™4 (catalog number 11818-010), andpENTR™11 (catalog number 11819-018).

[1053] In some embodiments, the present invention encompasses theexpression of fusion polypeptides comprising all or a portion of a humanpolypeptide. One suitable source of nucleic acid molecules encodinghuman polypeptides is the Ultimate™ Human ORF (hORF) Clone collectionavailable from Invitrogen Corporation, Carlsbad, Calif. To express ahuman gene of interest from pcDNA™6.2/V5-DEST or pcDNA™6.2/GFP-DEST, anUltimate™ Human ORF (hORF) Clone available from Invitrogen Corporation,Carlsbad, Calif. can be used. Each Ultimate™ hORF Clone is afully-sequenced clone provided in a GATEWAY® entry vector that isready-to-use in an LR recombination reaction with pcDNA™6.2/V5-DEST orpcDNA™6.2/GFP-DEST. In addition, each Ultimate™ hORF Clone contains aTAG stop codon, making it fully compatible for use in the Tag-on-Demand™System. For more information about the Ultimate™ hORF Clones available,see the Invitrogen Corporation, Carlsbad, Calif. Web site or contactInvitrogen Corporation, Carlsbad, Calif.

[1054] When generating an entry clone, a nucleic acid sequence encodinga polypeptide of interest in the entry clone must contain an ATGinitiation codon in the context of a Kozak consensus sequence for properinitiation of translation in mammalian cells as discussed above.

[1055] To enable expression of both a native and C-terminally-taggedrecombinant polypeptide of interest from pcDNA™6.2/V5-DEST orpcDNA™6.2/GFP-DEST using the Tag-on-Demand™ System, the gene of interestin the entry clone may contain a stop codon. This stop codon may beencoded by the nucleotides, TAG. In addition, the gene should be inframe with the C-terminal tag after recombination. Those skilled in theart will appreciate that other stop codons can be similarly used byconstructing a vector expressing a suppressor tRNA that recognizes theother stop codons.

[1056] The recombination region of pcDNA™6.2/V5-DEST andpcDNA6.2/GFP-DEST are provided as FIGS. 61A and 61B respectively. InFIG. 61A, shaded regions correspond to those DNA sequences transferredfrom the entry clone into the pcDNA™6.2/V5-DEST vector by recombination.Non-shaded regions are derived from the pcDNA™6.2/V5-DEST vector. Thesequences encoded by the gene of interest are boxed. To facilitate usewith the Tag-on-Demand™ System, a gene of interest must contain a TAGstop codon and be in-frame with the C-terminal tag. Bases 918 and 3161of the pcDNA™6.2/V5-DEST sequence are marked. Note that TAA and TGA stopcodons are included downstream of the V5 epitope to allow translationtermination in the Tag-on-Demand™ System. In FIG. 61B, the recombinationregion of the expression clone resulting from pcDNA™6.2/GFP-DEST× entryclone is shown. The shaded regions correspond to those DNA sequencestransferred from the entry clone into the pcDNA™6.2/GFP-DEST vector byrecombination. Non-shaded regions are derived from thepcDNA™6.2/GFP-DEST vector. The sequences encoded by the gene of interestare boxed. To facilitate use with the Tag-on-Demand™ System, the gene ofinterest should contain a TAG stop codon. Bases 918 and 3161 of thepcDNA™6.2/GFP-DEST sequence are marked. TAA and TGA stop codons areincluded downstream of the GFP gene to allow translation termination inthe Tag-on-Demand™ System (not shown).

[1057] To generate an expression clone: an LR recombination reactionusing the attL-containing entry clone and the attR-containingpcDNA™6.2/V5-DEST or pcDNA™6.2/GFP-DEST vector may be performed. Boththe entry clone and the destination vector may be supercoiled or linear.After the LR reaction has been performed, all or a portion of thereaction mixture may be used to transform a suitable E. coli host. Theexpression clones can be selected for using ampicillin and/orblasticidin.

[1058] The pcDNA™6.2/V5-DEST and pcDNA™6.2/GFP-DEST vectors are suppliedas supercoiled plasmids. Although the GATEWAY® Technology manual haspreviously recommended using a linearized destination vector for moreefficient recombination, it has been found that linearization ofpcDNA™6.2/V5-DEST and pcDNA™6.2/GFP-DEST is not required to obtainoptimal results for any downstream application.

[1059] Nucleic acid molecules of the invention, (e.g., destinationvectors) may be lyophilized for long term storage. Lyophilized plasmidsmay be resuspended in a suitable buffer (e.g., TE, pH 8.0). In someembodiments, the vectors may be lyophilized in a buffer (e.g., TE, pH8.0) and may be resuspended by the addition of sterile water. A suitableconcentration for solutions of nucleic acid molecules to be used in thepractice of the invention is about 150 ng/μl although otherconcentrations may be used.

[1060] In some embodiments, nucleic acid molecules of the invention maybe propagated in suitable host cells. To propagate and maintain thepcDNA™6.2/V5-DEST or pcDNA™6.2/GFP-DEST vectors, Library Efficiency®DB3.1™ Competent Cells (Invitrogen Corporation, Carlsbad, Calif. Catalogno. 11782-018) can be used. The DB3.1™ E. coli strain is resistant toCcdB effects and can support the propagation of plasmids containing theccdB gene. To maintain integrity of the vector, select for transformantsin media containing 50-100 μg/ml ampicillin and 15-30 μg/mlchloramphenicol. General E. coli cloning strains including TOP10 or DH5αshould not be used for propagation and maintenance as these strains aresensitive to CcdB effects.

[1061] Once an entry clone containing a gene of interest has beenprepared, perform an LR recombination reaction between the entry cloneand pcDNA™6.2/V5-DEST or pcDNA™6.2/GFP-DEST, and transform the reactionmixture into a suitable E. coli host. A negative control (no entryvector) is recommended to help evaluate results. Any recA, endA E. colistrain including TOP10, DH5α™, or equivalent for transformation can beused. Do not transform the LR reaction mixture into E. coli strains thatcontain the F′ episome (e.g., TOP10 F′). These strains contain the ccdAgene and will prevent negative selection with the ccdb gene.

[1062] The pcDNA™6.2/V5-DEST and pcDNA™6.2/GFP-DEST vectors contain theampicillin and Blasticidin resistance genes to allow selection of E.coli transformants using ampicillin or Blasticidin, respectively. Toselect for transformants using Blasticidin, use Low Salt LB agar platescontaining 100 μg/ml Blasticidin. For Blasticidin to be active, the saltconcentration of the medium must remain low (<90 mM) and the pH must be7.0. Low salt plates may be prepared by mixing 10 g Tryptone, 5 g NaCl,5 g Yeast Extract and adding deionized, distilled water to 950 ml.Adjust pH to 7.0 with 1 N NaOH. Bring the volume up to 1 liter. Forplates, add 15 g/L agar. before autoclaving.

[1063] Autoclave on liquid cycle at 15 psi and 121° C. for 20 minutes.Allow the medium to cool to at least 55° C. before adding theblasticidin to 100 μg/ml final concentration. Store plates at +4° C. inthe dark. Plates containing blasticidin are stable for up to 2 weeks.Blasticidin is available from Invitrogen Corporation, Carlsbad, Calif.

[1064] An LR recombination reaction may be performed with purifiedplasmid DNA of an entry clone (50-150 ng/μl in TE, pH 8.0);pcDNA™6.2/V5-DEST or pcDNA™6.2/GFP-DEST vector (150 ng/μl in TE, pH8.0); LR Clonase™ enzyme mix (Invitrogen, Catalog no. 11791-019; keep at−80° C. until immediately before use); 5× LR Clonase™ Reaction Buffer(supplied with the LR Clonase™ enzyme mix); TE Buffer, pH 8.0 (10 mMTris-HCl, pH 8.0, 1 mM EDTA); 2 μg/μl Proteinase K solution (suppliedwith the LR Clonase™ enzyme mix; thaw and keep on ice until use); anappropriate competent E. coli host and growth media for expression; SOCMedium; and selective plates (e.g., LB agar plates containing 100 μg/mlampicillin or Low Salt LB plates containing 100 82 g/ml Blasticidin).

[1065] Add the following components to 1.5 ml microcentrifuge tubes atroom temperature and mix. Component Sample Negative Control Entry clone(100-300 ng/reaction) 1-10 μl — Destination vector (300 ng/reaction) 2μl 2 μl 5 × LR Clonase ™ Reaction Buffer 4 μl 4 μl TE Buffer, pH 8.0 to16 μl 10 μl

[1066] Remove the LR Clonase™ enzyme mix from −80° C. and thaw on ice(˜2 minutes). Vortex the LR Clonase™ enzyme mix briefly twice (2 secondseach time). To each sample above, add 4 μl of LR Clonase™ enzyme mix.Mix well by pipetting up and down. Return LR Clonase™ enzyme mix to −80°C. immediately after use. Incubate reactions at 25° C. for 1 hour.Extending the incubation time to 18 hours typically yields morecolonies. Add 2 μl of the Proteinase K solution to each reaction.Incubate for 10 minutes at 37° C. Transform 1 μl of the LR recombinationreaction into a suitable E. coli host (follow the manufacturer'sinstructions) and select for expression clones. The LR reaction may bestored at −20° C. for up to 1 week before transformation, if desired.

[1067] If E. coli cells with a transformation efficiency of 1×10⁸ cfu/mgare used, the LR reaction should give approximately >5,000 colonies ifthe entire transformation is plated.

[1068] The ccdB gene mutates at a very low frequency, resulting in avery low number of false positives. True expression clones will beampicillin-resistant and chloramphenicol-sensitive. Transformantscontaining a plasmid with a mutated ccdB gene will be ampicillin- andchloramphenicol-resistant. To check a putative expression clone, testfor growth on LB plates containing 30 μg/ml chloramphenicol. A trueexpression clone should not grow in the presence of chloramphenicol.

[1069] To confirm that a gene of interest is in the correct orientationand in frame with the C-terminal fusion tag, the expression constructcan be sequenced. The following primers can be used to sequence anexpression construct. FIGS. 61A and 61B provide the location of theprimer binding sites in each vector. For sequencing thepcDNA™6.2/V5-DEST vector, an oligonucleotide that binds to the T7promoter/priming site (e.g., 5′-TAATACGACTCACTATAGGG-3′) and anoligonucleotide that binds to the V5(C-term) reverse priming site (e.g.,5′-ACCGAGGAGAGGGTTAGGGAT-3′) can be used. To sequence thepcDNA™6.2/GFP-DEST vector, an oligonucleotide that binds to the T7promoter/priming site (e.g., 5′-TAATACGACTCACTATAGGG-3′) and anoligonucleotide that binds to the GFP reverse priming site (e.g.,5′-GGGTAAGCTTTCCGTATGTAGC-3′) can be used.

[1070] Once an expression clone has been prepared, plasmid DNA fortransfection may be prepared. Plasmid DNA for transfection intoeukaryotic cells must be very clean and free from phenol and sodiumchloride. Contaminants will kill the cells, and salt will interfere withlipid complexing, decreasing transfection efficiency. Plasmid DNA can beisolated using the S.N.A.P.™ MidiPrep Kit (Invitrogen Corporation,Carlsbad, Calif. Catalog no. K1910-01) or CsCl gradient centrifugation.

[1071] For established cell lines (e.g., COS, HeLa), consult originalreferences or the supplier of the cell line for the optimal method oftransfection. It is recommended that the protocol developed forindividual cell lines be followed. Factors that may influencetransfection efficiencies include medium requirements, when to pass thecells, and at what dilution to split the cells. Further information isprovided in Current Protocols in Molecular Biology (Ausubel, F. M., etal., Current Protocols in Molecular Biology, Greene PublishingAssociates and Wiley-Interscience, New York (1994)).

[1072] Methods for transfection include calcium phosphate (Chen, C., andOkayama, H., Mol. Cell. Biol. 7:2745-2752 (1987); Wigler, M., et al.,Cell 11:223-232 (1977)), lipid-mediated (Felgner, P. L., et al., Proc.West. Pharmacol. Soc. 32:115-121 (1989); Felgner, P. L., and Ringold, G.M., Nature 337:387-388 (1989)) and electroporation (Chu, G., et al.,Nucleic Acids Res. 15:1311-1326 (1987); Shigekawa, K., and Dower, W. J.,BioTechniques 6:742-751 (1988)). If a cationic lipid-based reagent fortransfection is used, one suitable reagent is Lipofectamine™ 2000Reagent available from Invitrogen Corporation, Carlsbad, Calif. (Catalogno. 11668-027). Other suitable transfection reagents may also be used.

[1073] pcDNA™6.2/V5-GW/p64^(TAG) or pcDNA™6.2/GFP-GW/p64^(TAG) isprovided as a positive control vector for mammalian cell transfectionand expression and may be used to optimize recombinant proteinexpression levels in a particular cell line. These vectors allowexpression of native or C-terminally-tagged recombinant human c-myc(p64) protein that may be detected by Western blot. If using thesevectors as expression controls, be aware that the p64 protein isnaturally associated with nucleolar structures and requires ionicdetergents (RIPA or SDS gel loading buffer) to adequately solubilize intotal cell lysates prior to western blot analysis.

[1074] To propagate and maintain each of the control plasmids resuspendthe vector in 10 μl sterile water to prepare a 1 μg/μl stock solutionand use the stock solution to transform a recA, endA E. coli strain likeTOP10, DH5α™, or equivalent. Transformants can be selected on LB agarplates containing 100 μg/ml ampicillin or Low Salt LB agar platescontaining 100 μg/ml Blasticidin. A glycerol stock of a transformantcontaining plasmid can be prepared for long-term storage.

[1075] The methods described herein (e.g., the Tag-on-Demand™ System)can be used to express both native and C-terminally-tagged recombinantpolypeptide in mammalian cells from the same pcDNA™6.2/V5-DEST orpcDNA™6.2/GFP-DEST expression construct. To use the Tag-on-Demand™System, add the Tag-on-Demand™ Suppressor Supernatant to mammalian cellsat a specified time

[1076] In some embodiments, particularly those in which an adenovirus isused to transduce a host cell in order to express a suppressor tRNA, thehost cell may be transduced with the adenovirus followed immediately bytransfection with the expression construct containing a sequence ofinterest encoding a polypeptide of interest. Embodiments of this typemay be used to quickly screen for expression (or localization, ifpossible) of a recombinant polypeptide or to screen for expression of alarge number of polypeptides. Embodiments of this type will be discussedin greater detail in the following example.

[1077] In some embodiments, it may be desirable to generate a stablecell line comprising a nucleic acid molecule encoding a polypeptide ofinterest. In embodiments of this type, a nucleic acid molecule encodinga suppressor tRNA may be introduced into the stable cell line to producefusion polypeptides comprising the polypeptide of interest fused to anadditional polypeptide sequence (e.g., a tag sequence, etc.). Forexample, a stable cell line may be transduced with an adenoviral vectorexpressing one or more suppressor tRNAs (e.g., the Tag-On-Demand™Suppressor Supernatant) to produce a C-terminally-tagged recombinantpolypeptide.

[1078] In some embodiments (e.g., the Tag-on-Demand™ SuppressorSupernatant), nucleic acid molecules of the invention may be purified,titered, replication-incompetent, recombinant adenoviruses containing ahuman tRNA^(TAG) suppressor. Transduction of the adenovirus intomammalian cells facilitates transient stop suppression at the TAG codonin a gene of interest, enabling production of C-terminally-taggedrecombinant polypeptide.

[1079] In some embodiments (e.g., the Tag-on-Demand™ SuppressorSupernatant), a nucleic acid molecules of the invention may berecombinant adenovirus that is deleted in the E1 region. Such anadenovirus is replication-incompetent in any mammalian cells that do notexpress the E1 proteins. Using such adenoviruses in 293 cells or in anycell line that expresses the adenovirus E1 gene (Graham, F. L., et al.,J. Gen. Virol. 36:59-74 (1977); Kozarsky, K. F., and Wilson, J. M.,Curr. Opin. Genet. Dev. 3:499-503 (1993); Krougliak, V., and Graham, F.L., Hum. Gene Ther. 6:1575-1586 (1995)) results in viral replication andwill lead to rapid death of the target cell within 1-2 days afterinfection.

[1080] Using methods of the invention, fusion polypeptides may beexpressed transiently or stably. To express a recombinant fusionpolypeptide transiently, nucleic acid molecules encoding the fusionpolypeptide of interest and encoding and nucleic acid molecules encodinga suppressor tRNA may be introduced into a host cell. One skilled in theart will appreciate that the sequences encoding the fusion polypeptideof interest and the suppressor tRNA may be on the same or differentnucleic acid molecules. In embodiments where an adenovirus is used toexpress a suppressor tRNA, cells may be transduced with the adenovirusand then transfected with the expression construct (i.e. nucleic acidmolecule encoding the fusion polypeptide).

[1081] To express a recombinant fusion polypeptide from a stable cellline, a stable cell line comprising a nucleic acid molecule encoding thefusion polypeptide of interest may be created using any standardtechnique or one or more of the techniques described herein (e.g., usinglentiviral vectors or transfecting the mammalian cell line with thepcDNA™6.2/V5-DEST or pcDNA™6.2/GFP-DEST expression construct, etc.). Anucleic acid molecule encoding a suppressor tRNA (e.g., an adenovirusexpressing a suppressor tRNA) may be introduced into the stable cellline to produce a fusion polypeptide.

[1082] In some embodiments, nucleic acid molecules of the invention(e.g., pcDNA™6.2/V5-DEST and pcDNA™6.2/GFP-DEST vectors) may contain oneor more selectable markers that may be used to select for stable celllines. In one embodiment, nucleic acid molecules of the invention maycontain the Blasticidin resistance gene to allow selection of stablecell lines. Some methods of the invention may entail creating stablecell lines by transfecting a construct into a mammalian cell line ofchoice and selecting for foci using Blasticidin. Methods of creatingstable cell lines may also comprise linearizing a nucleic acid moleculeof the invention (e.g., pcDNA™6.2V5-DEST or pcDNA™6.2/GFP-DESTexpression constructs) before transfecting them into a host cell. Whilelinearizing the vector may not improve the efficiency of transfection,it increases the chances that the vector does not integrate in a waythat disrupts elements necessary for expression in mammalian cells.Linearizing may comprise digesting the construct with a restrictionenzyme that cuts at a unique site that is not located within a criticalelement or within the gene of interest.

[1083] In some embodiments, methods of generating a stable cell lineexpressing a polypeptide of interest may comprise determining theminimum concentration of Blasticidin required to kill the untransfectedhost cell line by performing a kill curve experiment using any one ofthe protocols described herein. Typically, concentrations ranging from2.5 to 10 μg/ml Blasticidin are sufficient to kill most untransfectedmammalian cell lines.

[1084] Once the appropriate Blasticidin concentration to use forselection has been determined, a stable cell line expressing a fusionpolypeptide of interest (e.g., a pcDNA™6.2/V5-DEST or pcDNA™6.2/GFP-DESTconstruct) can be generated. Methods of creating a stable cell line maycomprise transfecting a mammalian cell line of interest with a nucleicacid molecule of the invention (e.g., a pcDNA™6.2/V5-DEST orpcDNA™6.2/GFP-DEST construct) using any transfection method of choiceand selecting a stable cell line. Selecting may comprise 24 hours aftertransfection, washing the cells and adding fresh growth medium. 48 hoursafter transfection, splitting the cells into fresh growth medium suchthat they are no more than 25% confluent. If the cells are too dense,the antibiotic will not kill the cells. Antibiotics work best onactively dividing cells. Selecting may further comprise incubating thecells at 37° C. for 2-3 hours until they have attached to the culturedish, removing the growth medium and replacing with fresh growth mediumcontaining Blasticidin at the predetermined concentration required forthe cell line. Methods of creating a stable cell line may also comprisefeeding the cells with selective media every 3-4 days untilBlasticidin-resistant colonies can be identified. Pick at least 5Blasticidin-resistant colonies and expand them to assay for recombinantpolypeptide expression.

[1085] Methods of the invention may comprise detecting a fusionpolypeptide of the invention. For example, V5 fusion polypeptidesexpressed from pcDNA™6.2/V5-DEST can be detected using Western blot,immunofluorescence, or a functional assay specific the polypeptide ofinterest. A time course of expression may be prepared to optimizeexpression of the recombinant polypeptide (e.g. 24, 48, 72 hours, etc.).Anti-V5 Antibodies are available from Invitrogen Corporation, Carlsbad,Calif. and can be used to detect V5-tagged recombinant fusionpolypeptides: For Western blot analysis, the Anti-V5-HorseradishPeroxidase (HRP) Antibody or the Anti-V5-Alkaline Phosphatase (AP)Antibody may be used for detection. For immunofluorescence, theAnti-V5-Fluorescein Isothiocyanate (FITC) Antibody can be used fordetection.

[1086] Methods of detecting a fusion polypeptide may comprise performinga Western blot. Such a method may comprise preparing a cell lysate fromtransfected cells. Any suitable protocol for preparing a cell lysateknown to those skilled in the art may be used. Preparing a cell lysatemay comprise washing cell monolayers (e.g., ˜5×10⁵ to 1×10⁶ cells may bewashed once with Phosphate-Buffered Saline, PBS, Invitrogen Corporation,Carlsbad, Calif., Catalog no. 10010-023). Preparing a cell lysate mayfurther comprise scraping cells into a buffer and centrifuging thecells. For example, cells may be scraped into 1 ml PBS and cells may becentrifuged at 1500×g for 5 minutes to form a cell pellet. Methods ofpreparing a cell lysate may comprise re-suspending a cell pellet in alysis buffer. For example, cells may be re-suspended in 50 μl Cell LysisBuffer (e.g., 50 mM Tris, pH 7.8, 150 mM NaCl, 1% Nonidet P-40). Othercell lysis buffers known to those skilled in the art are also suitable.Re-suspending may comprise mixing (e.g., vortexing) the cell pellet inthe lysis buffer to form a cell suspension and incubating the cellsuspension (fore example, at 37° C. for 10 minutes) under conditionssuitable to lyse the cells. Cells may be lysed at room temperature or onice if degradation of polypeptide is a potential problem. Methods ofpreparing a cell lysate may further comprise centrifuging the celllysate, for example, at 10,000×g for 10 minutes at +4° C. to pelletnuclei and transferring the supernatant to a fresh tube.

[1087] Lysates prepared according to the invention may be furtheranalyzed using techniques well known in the art, for example, lysatesmay be assayed for protein concentration. Those skilled in the art willappreciate that protein assays utilizing Coomassie Blue or other dyesshould not be used if the lysis buffer comprises NP-40 since NP-40interferes with the binding of the dye with the protein.

[1088] Methods of performing a Western blot may comprise mixing analiquot of a cell lysate with an SDS-PAGE. For example, SDS-PAGE samplebuffer can be added to cell lysate to from a mixture and the mixture maybe boiled, for example, for 5 minutes. An amount of the mixturecomprising about 20 μg of protein may be loaded onto an SDS-PAGE gel andelectrophoresed. One skilled in the art can select the appropriateconcentration of acrylamide to be used to prepare the gel based upon theexpected size of the fusion polypeptide.

[1089] One skilled in the art will recognize that a C-terminal tagcontaining the attB2 site and the V5 epitope will add approximately 4kDa to the polypeptide of interest. Fusion polypeptides of the inventionmay also comprise additional amino acids located between the polypeptideof interest and the additional polypeptide sequence (e.g., a tagsequence such as the V5 epitope).

[1090] In some embodiments, methods of the invention may comprisedetecting the presence of a fusion polypeptide comprising all or aportion of the p64 polypeptide. Methods of this type (e.g., fusionpolypeptides expressed from the pcDNA™6.2/V5-GW/p64TAG control), mayutilize any suitable detection means, for example, any of the Anti-V5Antibodies and/or anti-myc antibodies discussed above. Methods ofpreparing a cell lysate from a cell expressing a fusion polypeptidecomprising all or a portion of p64 may comprise the use of harsherextraction conditions since procedures using NP-40 lysis are noteffective in releasing p64 protein. Since p64 is localized in thenucleoli, harsher lysis procedures using RIPA or SDS-PAGE sample bufferare required to adequately solubilize p64 in total cell lysates. Methodsof this type may comprise washing cell monolayers, for example, oncewith Phosphate-Buffered Saline (PBS, Invitrogen Corporation, Carlsbad,Calif. Catalog no. 10010-023). Methods may further comprise add 1×SDS-PAGE Sample Buffer to each well containing cells. 1× SDS-PAGE buffercan be prepared by mixing 2.5 ml 0.5 M Tris-HCl, pH 6.8, 2 ml ofglycerol (100%), 0.4 ml of β-mercaptoethanol, 0.02 g Bromophenol Blue,0.4 g SDS and enough sterile water to bring the volume to 20 ml. For a24-well plate, use 100 μl of 1× SDS-PAGE Sample Buffer per well. Methodsmay further comprise removing the cells from the plate, for example, apipette tip may be used to loosen lysed cells from plate. Lysed cellsmay be transferred to a 1.5 ml microcentrifuge tube. Lysates preparedaccording to this method are typically viscous. Methods may furthercomprise heating samples, for example, at 70° C. for 10 minutes andmixing samples, for example, by vortexing every few minutes and brieflycentrifuging the sample.

[1091] Methods may further comprise loading an aliquot of the celllysate, for example, 5 μl of cell lysate, onto an SDS-PAGE gel andelectrophoresing. One skilled in the art will appreciate that theV5-tagged p64^(TAG) protein has a molecular weight of approximately 53kDa.

[1092] To detect the polypeptides expressed from as cycle-3 GFP fusionpolypeptides from pcDNA™6.2/GFP-DEST, fluorescence, Western blotanalysis, or a functional assay specific for the polypeptide of interestmay be used. A time course may be prepared to optimize expression of therecombinant polypeptide (e.g. 24, 48, 72 hours, etc.). Any suitabletechnique, including those discussed herein, may be used to evaluateexpression.

[1093] Cycle-3 GFP fusion polypeptides may be detected in vivo usingfluorescence microscopy. The CMV promoter used to control expression ofthe cycle-3 GFP fusion polypeptide from pcDNA™6.2/GFP-DEST is a strongpromoter and typically cycle-3 GFP fluorescence may be detected about 24hours after transfection or transduction.

[1094] Methods of the invention may comprise methods of detectingfluorescent cells. In the practice of such methods, it is important topick the best filter set to optimize detection. The primary excitationpeak of cycle-3 GFP is at 395 nm. There is a secondary excitation peakat 478 nm. Excitation at either of these wavelengths yields afluorescent emission peak with a maximum at 507 nm. Note that thequantum yield can vary as much as 5- to 10-fold depending on thewavelength of light that is used to excite the GFP fluorophore.

[1095] Use of the best filter set will insure that the optimal regionsof the cycle-3 GFP spectra are excited and passed. Suitable filter setsinclude those designed to detect fluorescence from wild-type GFP (e.g.,Omega Optical XF76 filter; see www.omegafilters.com). FITC filter setsmay be used to detect cycle-3 GFP fluorescence, but note that these arenot optimal and fluorescent signal may be weaker. For example, a FITCfilter set may excite cycle-3 GFP with light from 460 to 490 nm,covering the secondary excitation peak and pass light from 515 to 550nm. A set of this type may allow detection of most but not all of thecycle-3 GFP fluorescence.

[1096] Most tissue culture media fluoresce because of the presence ofriboflavin (Zylka, M. J., and Schnapp, B. J., BioTechniques 21:220-226(1996)) and may interfere with detection of cycle-3 GFP fluorescence.Medium can be removed and replaced with Phosphate-Buffered Saline (PBS,Invitrogen Corporation, Carlsbad, Calif., Catalog no. 10010-023) duringthe assay to alleviate this problem. If cells will be cultured furtherafter assaying, remove the PBS and replace with fresh growth mediumprior to re-incubation.

[1097] To detect expression of a cycle-3 GFP fusion polypeptide byWestern blot, an antibody to the polypeptide of interest or an antibodyto cycle-3 GFP may be used. GFP Antiserum is available separately fromInvitrogen Corporation, Carlsbad, Calif. (Catalog no. R970-01) fordetection. The GFP Antiserum is a purified, polyclonal rabbit antiserumraised against recombinant cycle-3 GFP, and can detect both cycle-3 GFPand wild-type GFP protein.

[1098] The C-terminal tag containing the attB2 site and cycle-3 GFP willadd approximately 28.3 kDa to the size of the fusion polypeptide. Fusionpolypeptides of the invention may further comprise additional aminoacids located between the polypeptide of interest and cycle-3 GFP.

Example 16

[1099] In some embodiments, the present invention provides materials andmethods for the expression of fusion polypeptides. In one aspect, thesame nucleic acid molecule is used to express a polypeptide of interestand a fusion polypeptide comprising the polypeptide of interest. In someaspects, this is accomplished by introducing into a host cell a nucleicacid molecule encoding a fusion polypeptide comprising a polypeptide ofinterest in the same reading frame as an additional polypeptidesequence. Typically, the nucleic acid molecule encoding the fusionpolypeptide may comprise one or more stop codons, one of which may belocated between the portion of the nucleic acid sequence encoding thepolypeptide of interest and the portion of the nucleic acid sequenceencoding the additional polypeptide sequence. In the presence of anucleic acid molecule expressing one or more nucleic acid sequencesencoding suppressor tRNA molecules, the stop codon between the twopolypeptide sequences is suppressed and a fusion polypeptide isexpressed.

[1100] Thus, in one aspect, the present invention comprises nucleic acidmolecules (and/or compositions comprising such molecules) from whichtRNA molecules (e.g., suppressor tRNA molecules) can be expressed.Nucleic acid molecules from which tRNA molecules can be expressed may beany type nucleic acid molecule known to those skilled in the art, forexample, plasmids, linear nucleic acid molecules, viruses and the like.In a particular embodiment, the present invention provides a virus(e.g., an adenovirus, a lentivirus, a baculovirus etc.) from which atRNA molecule may be expressed. In a specific embodiment, the presentinvention provides an adenovirus from which one or more tRNA moleculemay be expressed.

[1101] In one embodiment, the present invention provides an adenovirusthat expresses one or more suppressor tRNA molecules. One non-limitingexample of such an adenovirus can be found in the Tag-On-Demand™ Systemcommercially available from Invitrogen Corporation, Carlsbad, Calif.catalog number K400-01. Methods of the invention may employ anadenoviral-based stop suppression technology to allow expression of anuntagged (i.e. native) or C-terminally-tagged recombinant polypeptide ofinterest in host cells from a single expression vector. In someembodiments, nucleic acid molecules of the invention may includeTag-On-Demand™ GATEWAY® vectors and/or other vectors, which may be usedto generate an expression construct.

[1102] In one aspect, materials and methods of the present invention maybe used to facilitate transient expression of a C-terminally-taggedrecombinant polypeptide of interest in host cells (e.g., mammaliancells). Materials and methods of the invention may be used to provide ameans to easily detect the expression or localization of a recombinantpolypeptide(s) for which there is no specific antibody available. Thismay be useful, for example, in that once tagged recombinant polypeptideexpression is verified, native polypeptide expression experiments may beperformed with the same construct.

[1103] In some aspects, the present invention uses adenovirus as adelivery vehicle, enabling efficient delivery of suppressor tRNAs to alarge variety of host cell types (e.g., mammalian cell types).Typically, in methods of the invention, suppressor tRNAs may bedelivered transiently to cells to minimize toxicity.

[1104] In some aspects of the invention, methods of the invention may beused for high-throughput applications including rapid screening of alarge number of genes for expression in a particular cell type.

[1105] In some embodiments, materials and methods of the invention maybe used to transiently express C-terminally-tagged and nativerecombinant polypeptides in mammalian cells from a single expressionconstruct. Suppressor tRNAs that function in mammalian cells have beendescribed (see Capone, et al. (1985) EMBO J. 4, 213-221).

[1106] In one aspect, the present invention provides nucleic acidmolecules (e.g., mammalian expression vectors) into which the a nucleicacid sequence encoding a polypeptide of interest will be cloned.Preferably, a nucleic acid sequence encoding a polypeptide of interestmay be cloned into a nucleic acid molecule of the invention (e.gpcDNA™6.2/V5-DEST or pcDNA™6.2/GFP-DEST) in a configuration that iscompatible with expression of C-terminally-tagged recombinantpolypeptide by suppression of one or more stop codons.

[1107] In another aspect, the present invention provides nucleic acidmolecules (e.g., replication-incompetent adenoviruses) comprising anucleic acid sequence from which a suppressor tRNA can be expressed(e.g., the human tRNA^(ser) suppressor gene). In some embodiments, asuppressor tRNA may be a tRNA mutated to recognize one or more stopcodons, for example, the TAG (amber stop) codon, and decode it as aserine. Nucleic acid molecules according to this aspect of the inventionmay be introduced into host cells to provide a transient source of thetRNA^(ser) suppressor. If the expression construct encoding a gene ofinterest with a TAG stop codon is present in the host cells, the stopcodon will be translated as serine, allowing translation to continuethrough any downstream reading frame (i.e. C-terminal tag). This resultsin production of a C-terminally-tagged fusion polypeptide.

[1108] In one aspect, a nucleic acid molecule from which a suppressortRNA molecule may be expressed may be a recombinant adenovirus and maybe constructed as follows. A vector containing the gene encoding thetRNA^(ser) gene with its native promoter and terminator may be obtained,for example, from Dr. Uttam RajBhandary at the Massachusetts Instituteof Technology. This tRNA^(ser) gene has been mutated such that theanticodon recognizes the TAG (amber) stop codon, and is referred to asthe tRNA^(ser) suppressor gene (see Capone, et al. (1985)). ThetRNA^(ser) suppressor gene may be PCR amplified and TOPO® Cloned intothe pENTR/D-TOPO® vector available from Invitrogen Corporation,Carlsbad, Calif. (Catalog no. K2400-20) to generate a GATEWAY® entryclone. Using the entry clone above and a multimerization proceduredescribed in Buvoli et. al., 2000 (Buvoli, et al. (2000) Mol. Cell.Biol. 20, 3116-3124), a GATEWAY® entry clone containing 8 tandem copiesof the tRNA^(ser) suppressor gene can be generated. One such entry clonehas been constructed and is named pENTR™-tRNA8^(TAG). ThepENTR™-tRNA8^(TAG) entry clone was recombined with Invitrogen'spAd/PL-DEST™ destination vector (Catalog no. V494-20) using the GATEWAY®LR recombination reaction to generate the adenoviral expression clone,pAd/GW-tRNA8^(TAG). The pAd/GW-tRNA8^(TAG) expression construct was usedin Invitrogen's ViraPower™ Adenoviral Expression System (Catalog no.K4940-00) to produce recombinant adenovirus, which was CsCl-purified andtitered to generate the Tag-On-Demand™ Suppressor Supernatant.

[1109] In some embodiments, a nucleic acid molecule expressing asuppressor tRNA molecule may be a recombinant adenovirus, which may beused, for example, to deliver the tRNA^(ser) suppressor to host cells(e.g., mammalian cells). Although adenovirus has a very broad tropismand can be used to deliver the tRNA^(ser) suppressor to a large varietyof host cell lines and cell types, materials an methods of the inventionare not limited to those cells that can be transduced with adenovirus.Thus, materials and methods of the invention may be used with any hostcell line or type known to those skilled in the art.

[1110] In the practice of the invention, nucleic acid moleculesexpressing one or more suppressor tRNA molecules may be introduced intohost cells. When such nucleic acid molecules are introduced into hostcells, they may be introduced into from about 25% to about 100% of thecell population, or from about 25% to about 90%, from about 25% to about80%, from about 25% to about 70%, from about 25% to about 60%, fromabout 25% to about 50%, from about 50% to about 100%, from about 60% toabout 100%, from about 70% to about 100%, from about 80% to about 100%,from about 90% to about 100%, from about 50% to about 90%, from about50%, to about 80%, from about 50% to about 75%, from about 50% to about70%, or from about 50% to about 60%. In some embodiments, nucleic acidmolecules expressing one or more suppressor tRNAs may be adenovirusesand may transduce mammalian cells with extremely high efficiency,resulting in delivery of the tRNA^(ser) suppressor to nearly 100% ofmammalian cells.

[1111] In some embodiments, nucleic acid molecules expressing suppressortRNAs of the invention may not integrate into the host genome andexpression of the suppressor tRNA may be transient and only persist foras long as the nucleic acid molecule (e.g., viral genome) is present(typically 7-8 days after transduction). Typically, once an nucleic acidmolecule expressing a suppressor tRNA is introduced into host cells, thesuppressor tRNA is expressed within 24 hours.

[1112] In some embodiments, a nucleic acid molecule expressing asuppressor tRNA molecule may be a virus (e.g., adenovirus). As is knownin the art, viruses may possess the ability to bind to one or morereceptors that may be present on a cell surface. For example, adenovirusenters target cells by binding to the Coxsackie/Adenovirus Receptor(CAR) (see, Bergelson, et al. (1997) Science 275, 1320-1323) andinternalizing via integrin-mediated endocytosis (see, Russell, W. C.(2000) J. Gen. Virol. 81, 2573-2604). Once internalized, the recombinantadenovirus is actively transported to the nucleus, and begins to expressthe suppressor genes. Thus, when the nucleic acid molecule expressing asuppressor tRNA is an adenovirus, the host cell line should contain CAR.Most mammalian cell types express CAR, but levels vary. Depending on theamount of the CAR expressed in a specific target cell line, transductionefficiencies may vary when an adenovirus is used to express a suppressortRNA. One skilled in the art will appreciate that other viruses may beused to express suppressor tRNAs in the practice of the invention, forexample, vaccinia virus, herpes virus, adeno-associated virus,baculovirus, retroviruses (e.g., lentivirus), plant viruses (e.g.,tobacco mosaic virus, cauliflower mosaic virus, etc.), negative strandedRNA viruses (e.g., Sendai virus, etc.), positive stranded RNA viruses(eg., alphaviruses, etc.). One skilled in the art can readily select anappropriate virus to infect any desired type of target cell based on theknown tropisms of specific viruses for specific cell types.

[1113] In some embodiments, nucleic acid molecules expressing asuppressor tRNA of the invention may be adenoviruses. Adenoviruses foruse in this aspect of the invention may have one or more deletions inthe adenoviral genome compared to a wild-type adenoviral genome (e.g.,Ad2, Ad5, etc.). For example, an adenovirus for use in the invention maybe deleted in the E1 and/or E3 regions. In some embodiments, the entireE1 and E3 regions may be deleted. Such viruses may bereplication-incompetent when transduced into mammalian cells that do notexpress the E1a or E1b proteins (see, Graham, et al. (1977) J. Gen.Virol. 36, 59-74; Kozarsky and Wilson (1993) Curr. Opin. Genet. Dev. 3,499-503; and Krougliak and Graham (1995) Hum. Gene Ther. 6, 1575-1586).

[1114] As is known in the art, adenovirus does not integrate into thehost genome upon transduction. Because the virus isreplication-incompetent, the presence of the viral genome is transientand will eventually be diluted out as cell division occurs. Because theadenovirus is present transiently in mammalian cells, the production ofC-terminally-tagged polypeptide resulting from stop suppression is alsotransient. As levels of adenovirus decrease, levels ofC-terminally-tagged polypeptide produced decrease.

[1115] In some embodiments, viruses used to express suppressor tRNAs inaccordance with the invention may be replication-incompetent in the celltype in which they express the suppressor tRNA. Viruses for use in thisaspect of the invention may be screened for the presence of wild-typereplication-competent viruses using techniques known in the art. Forexample, a population of adenovirus for use in the present invention maybe screened for the presence of replication-competent adenovirus (RCA)contamination using a supernatant rescue assay (see, Dion, et al. (1996)J. Virol. Methods 56, 99-107) with a detection sensitivity of onewild-type RCA per 10⁹ recombinant adenovirus. In some embodiments, aviral preparation to be used to express one or more suppressor tRNAmolecules in accordance with the methods of the invention may contain nodetectable wild-type RCA.

[1116] In some embodiments, the present invention provides methods toexpress a C-terminally-tagged fusion polypeptide, comprising transducinga host cell with a virus expressing one or more suppressor tRNAmolecules, transfecting the transduced cells with one or more nucleicacid molecules encoding all or a portion of a fusion polypeptide, andincubating the host cell under conditions sufficient to express aC-terminally tagged fusion polypeptide. A schematic representation of anembodiment of this type is provided in FIG. 62. In another embodiment,the present invention provides methods to express a C-terminally taggedfusion polypeptide, comprising transducing a stable cell line comprisinga nucleic acid molecule encoding all or a portion of a fusionpolypeptide with a virus expressing one or more suppressor tRNAmolecules and incubating the transduced cell under conditions sufficientto express a C-terminally tagged fusion polypeptide. A schematicrepresentation of an embodiment of this type is shown in FIG. 63.

[1117] Methods of the invention may entail the use of stocks of viruses,for example, viruses expressing one or more suppressor tRNA molecules.As will be appreciated by those skilled in the art, stocks of virusesmay be stored at −80° C. In general, stocks stored under theseconditions are stable for at least 6 months. If a viral stock has beenstored at −80° C. for longer than 6 months, the tier of the stock may bedetermined using standard techniques as viral titers may decrease withlong-term storage. Viral stocks should not be repeatedly thawed andre-frozen as viral titers can decrease with more than 3 freeze/thawcycles.

[1118] One skilled in the art is aware that the handling of materialscontaining viruses should be performed following the applicable Federaland institutional guidelines for working with potentially hazardousorganisms. For example, all manipulations should be performed within acertified biosafety cabinet, all media containing virus should betreated with bleach, all material that comes into contact with virus(e.g., pipettes, pipette tips, and other tissue culture supplies) shouldbe treated with bleach or disposed of as biohazardous waste, and personshandling material containing virus should wear appropriate safetyclothing (e.g., gloves, a laboratory coat, and safety glasses orgoggles).

[1119] In some embodiments, methods of the invention may be used tocreate a nucleic acid molecule encoding a fusion polypeptide. Accordingto one aspect of the invention, a nucleic acid molecule encoding afusion polypeptide may be constructed by combining a first nucleic acidmolecule having a first nucleic acid sequence encoding a polypeptidesequence (e.g., a polypeptide of interest) with a second nucleic acidmolecule having a second nucleic acid sequence encoding an additionalpolypeptide sequence (e.g., a polypeptide tag sequence). A nucleic acidmolecule encoding a polypeptide of interest should contain an ATGinitiation codon in the context of a Kozak consensus sequence for properinitiation of translation in mammalian cells (Kozak, 1987; Kozak, 1991;Kozak, 1990). An example of a Kozak consensus sequence is (G/A)NNATGG,where the ATG initiation codon is underlined. Other sequences arepossible, but the G or A at position −3 and G at position +4 are themost critical for function (shown in bold).

[1120] Typically, a nucleic acid molecule encoding a polypeptide ofinterest will contain a stop codon, for example, encoded by thenucleotides, TAG, TAA or TGA. One skilled in the art will appreciatethat an appropriate suppressor tRNA (i.e., with an anti-codon thatcorresponds to the stop codon) must be used.

[1121] One skilled in the art will appreciate that, after joining of thefirst and second nucleic acid molecules to produce a nucleic acidmolecule encoding a fusion polypeptide, the sequence encoding thepolypeptide of interest must be in the same reading frame as theadditional polypeptide sequence.

[1122] Second nucleic acid molecules encoding an additional polypeptidesequence will typically comprise one or more stop codons after thesequence encoding the additional polypeptide sequence. In general, thestop codon on the second nucleic acid molecule will be different fromthe stop codon on the first nucleic acid molecule.

[1123] A wide variety of nucleic acid molecules are suitable for use assecond nucleic acid molecules in accordance with the present invention.Non-limiting examples of such nucleic acid molecules include vectorscommercially available from Invitrogen Corporation, Carlsbad, Calif.Examples of such vectors are provide with their Invitrogen Corporation,Carlsbad, Calif. catalog number in parenthesis. Such vectors include,but are not limited to, pLenti4/V5-DEST™ (K4980-00), pLenti6/V5-DEST™(K4950-00), pLenti6/UbC/V5-DEST™ (K4990-00), pLenti6/V5-D-TOPO®(K4960-00), and pAd/CMV/V5-DEST (K4930-00), which may be used for viralexpression; pcDNA5/FRT/V5-His-TOPO® (K6020-01), pSecTag/FRT/V5-His-TOPO®(K6025-01), pEF5/FRT/V5-DEST™ (V6020-20), and pEF5/FRT/V5-D-TOPO®(K6035-01), which may be used for expression from a specific genomiclocus using the Flp-In™ System; pcDNA™4/TO/myc-His (K1030-01),pGene/V5-His (K1060-01), which may be used for inducible expression;pcDNA™6.2/V5-DEST (K420-01), pcDNA™3.2/V5-DEST (12489-019),pcDNA™-DEST40 (12274-015), pcDNA6.2/V5-GW/D-TOPO® (K2460-20),pcDNA3.2/V5-GW/D-TOPO® (K2440-20), pcDNA3.1D/V5-His-TOPO® (K4900-01),pcDNA™3.1/V5-His-TOPO® (K4800-01), pcDNA™3.1/V5-His (V810-20),pcDNA™3.1/myc-His (V800-20), pcDNA™3.1(−)/myc-His (V855-20),pcDNA™4/V5-His (V861-20), pcDNA™4/myc-His (V863-20), pcDNA™6/V5-His(V220-20), and pcDNA™6/myc-His (V221-20), which may be used forconstitutive expression from the CMV promoter; pEF6/V5-His-TOPO®(K9610-20), pEF1/V5-His (V920-20), pEF1/myc-His (V921-20), pEF4/V5-His(V941-20), pEF4/myc-His (V942-20), pEF6/V5-His (V961-20), andpEF6/myc-His (V962-20), which may be used for constitutive expressionfrom the EF-1α promoter; pUB6/V5-His (V250-20), which may be used forconstitutive expression from the UbC promoter; pSecTag2 (V900-20), andpSecTag2/Hygro (V910-20), which may be used for constitutive secretedexpression; and pcDNA™6.2/GFP-DEST (K410-01), pcDNA™-DEST47 (12281-010),and pcDNA3.1/CT-GFP-TOPO® (K4820-01), which may be used for fusion tothe GFP reporter gene.

[1124] A variety of factors may be optimized to produce fusionpolypeptides according to the methods of the invention. Factors include,but are not limited to, characteristics of the host cell line; thehealth of the cells and experimental cell culture conditions; thetransfection method used to introduce nucleic acid molecules into thehost cell line; the transduction procedure used; and the amount ofnucleic acid encoding a suppressor tRNA introduced into the host cells(e.g., multiplicity of infection when the nucleic acid molecule encodinga suppressor tRNA is a virus such as an adenovirus).

[1125] In some embodiments, a fusion protein of the invention may beexpressed in any host cell type known to those skilled in the art. Insome embodiments, a host cell line may be a mammalian host cell line.When an adenovirus is used in the practice of the invention to expressone or more suppressor tRNA molecules, a host cell line preferablyexpresses one or more receptors allowing efficient transduction of thecell line by the adenovirus. An example of a suitable receptor is theCoxsackie/Adenovirus Receptor (CAR) (see, Bergelson, et al. (1997)Science 275, 1320-1323). Most mammalian cell types express CAR, butlevels vary. One skilled in the art will appreciate that transductionefficiencies of cell lines will vary depending on the amount of the CARexpressed in a given cell line and can adjust either the multiplicity ofinfection and/or the cell line used as necessary for any particularapplication using routine experimentation.

[1126] In some embodiments, cells lines used in the practice of thepresent invention may not express viral proteins necessary forreplication of a virus used to introduce suppressor tRNAs into the hostcells. For example, when an adenovirus is sued to introduce suppressortRNAs into host cells, the host cells may not express the adenovirus E1proteins.

[1127] Typically, host cells used in the practice of the invention maybe amenable to efficient transfection. For example, it may be possibleto introduce a nucleic acid molecule encoding a fusion polypeptideinvention into a high percentage of cells using standard techniques. Forexample, using lipid-mediated transfection (for example, withLipofectamine 2000), it may be possible to introduce a nucleic acidmolecule encoding a fusion polypeptide of the invention into from about25% to about 100%, from about 25% to about 99%, from about 25% to about95%, from about 25% to about 80%, from about 25% to about 70%, fromabout 25% to about 60%, from about 25% to about 50%, from about 25% toabout 40%, from about 40% to about 95%, from about 50% to about 95%,from about 60% to about 90%, from about 75% to about 95%, or from about80% to about 95% of the cells of a given sample of cells (e.g., thecells in a well of a tissue culture plate). Examples of suitable celllines include, but are not limited to, COS-7, CHO-S, HeLa, HT1080, andBHK-21, primary rat hippocampal and cortical neurons.

[1128] In some embodiments, nucleic acid molecules encoding suppressortRNAs for use in the present invention may be adenoviruses. Suchadenoviruses may be deleted in the E1 region, rendering themreplication-incompetent in any cells that do not express the E1proteins. Typically methods of the invention are not performed in cellsthat express the adenovirus E1 protein (e.g., 293 cells or derivatives)as viral replication may occur in these cells, leading to rapid death ofthe target cell within 1-2 days after infection. In some instances itmay be desirable to practice methods of the invention in cellsexpressing the adenovirus E1 protein.

[1129] One skilled in the art will appreciate that the health of thecells to be used in methods of the invention may affect the expressionof fusion polypeptide of the invention expressed in these cells. Ingeneral, in methods of the invention, cells should be healthy (i.e.exhibit>95% viability) at the time of plating. Poor quality cell stock(e.g. cells consistently allowed to become overgrown or confluent beforepassaging, growth media allowed to become yellow before re-feeding) cannegatively impact suppression efficiency and the amount of fusionpolypeptide expressed. Generally, freshly prepared media may be used inthe practice of methods of the invention.

[1130] Methods of the invention may entail introducing one or morenucleic acid molecules into one or more host cells. Any method of choicemay be used to transfect nucleic acid molecules into cells. Suitablemethods include, but are not limited to, calcium phosphate (see Chen andOkayama (1987); Wigler et al. (1977)), lipid-mediated (see, Felgner etal. (1989); Felgner and Ringold (1989)), and electroporation (see Chu,et al. (1987); Shigekawa and Dower (1988)). Suitable conditions (e.g.,reagents, incubation conditions, etc.) for introducing nucleic acidmolecules into any specific cell line may be determined by consultingpublished literature, consulting the provider of the cell line inquestion, and/or by routine experimentation. In some embodiments,methods of the invention may entail introducing one or more nucleic acidmolecules into one or more cells using lipid-mediated transfection witha suitable lipid reagent (e.g., a lipid reagent from InvitrogenCorporation, Carlsbad, Calif. such as a cationic lipid-based reagent,Lipofectamine™ 2000 Reagent).

[1131] In some embodiments, methods of the invention may compriseintroducing one or more nucleic acid molecules into one or more cellsusing Lipofectamine™ 2000 Reagent (see, Ciccarone, et al. (1999) Focus21, 54-55) a cationic lipid-based formulation designed for transfectionof nucleic acids into eukaryotic cells. Methods of this type maycomprise forming a complex comprising nucleic acid molecules andLipofectamine™ 2000 Reagent and contacting cells with the complexes inculture medium in the presence of serum. Such methods may not compriseremoval of complexes or medium change or addition followingtransfection. Alternatively, methods may comprise removal of complexesor medium change or addition following transfection, for example, at 4-6hours after contacting cells with the complexes.

[1132] In some embodiments, the present invention may include a methodof screening for expression of a polypeptide comprising introducing intoa host cell a nucleic acid molecule expressing a suppressor tRNA and anucleic acid molecule encoding the polypeptide; and detecting thepresent of the polypeptide. In some embodiments such methods may be usedto screen for expression or localization of the polypeptide or to screenfor expression of a large number of genes. Such methods may involve theuse of an adenovirus expressing a suppressor tRNA and may involvetransducing a host cell with the adenovirus and transfecting a nucleicacid molecule encoding the polypeptide. Typically, transfection of thenucleic acid molecule is done as soon as practical after transductionwith the adenoviruses. In some embodiments, the cells may be contactedwith a solution comprising the adenovirus and then nucleic acidmolecules (e.g., in complex with a transfection reagent) may be added tothe solution comprising the adenovirus. Optionally, a nucleic acidmolecule encoding a polypeptide of the invention may be introduced intoa host cell prior to transduction of the host cell with an adenovirusexpressing one or more suppressor tRNAs. One skilled in the art willappreciate that transducing a host cell with an adenovirus andsimultaneously (i.e. as soon as practically possible) transfecting withplasmid encoding a polypeptide of the invention can increaseplasmid-derived gene expression as well as reduce toxicity to the cell(see, Cotten, et al. (1992) Proc. Natl. Acad. Sci. USA 89, 6094-6098;Curiel, et al. (1991) Proc. Natl. Acad. Sci. USA 88, 8850-8854; Guy, etal. (1995) Mol. Biotechnol. 3, 237-248; Honda, et al. (1996) J. Virol.Methods 58, 41-51; and Merwin, et al. (1995) J. Immunol. Methods 186,257-266).

[1133] In some embodiments, it may be desirable to create a stable cellline comprising a nucleic acid molecule encoding a polypeptide of theinvention using standard techniques and to transduce the stable cellline with an adenovirus expressing one or more suppressor tRNAs.

[1134] In methods of the invention that comprise transducing a host cellwith a virus expressing one or more suppressor trans (e.g., anadenovirus), cells may be transduced with any desired amount of virus.For example, cells may be transduced with virus at a multiplicity ofinfection (MOI) of from about 0.1 to about 500, from about 0.25 to about500, from about 0.5 to about 500, from about 0.75 to about 500, fromabout 1 to about 500, from about 2 to about 500, from about 3 to about500, from about 4 to about 500, from about 5 to about 5000, from about10 to about 500, from about 25 to about 500, from about 50 to about 500,from about 75 to about 500, from about 100 to about 500, from about 200to about 500, from about 300 to about 500, from about 400 to about 500,from about 1 to about 250, from about 1 to about 200, from about 1 toabout 150, from about 1 to about 100, from about 1 to about 75, fromabout 1 to about 50, from about 1 to about 25, from about 1 to about 20,from about 1 to about 15, from about 1 to about 10, from about 1 toabout 5, from about 10 to about 400, from about 10 to about 300, fromabout 10 to about 200, from about 10 to about 100, from about 10 toabout 75, from about 10 to about 70, from about 10 to about 65, fromabout 10 to about 60 from about 10 to about 55, from about 10 to about50, from about 10 to about 45, from about 10 to about 40, from about 10to about 35, from about 10 to about 30, from about 10 to about 25, fromabout 10 to about 20, or from about 10 to about 15. Thus, cells may betransduced at an MOI of about 1, about 5, about 10, about 15, about 20,about 25, about 30, about 35, about 40, about 45, about 50 ,about 55,about 60, about 65, about 70, about 75, about 80, about 85, about 90,about 95, or about 100. MOI is defined as the number of virus particlesper cell and generally correlates with expression. Depending on the cellline used and the nature of the gene of interest, MOI may be varied tooptimize expression of a fusion polypeptide of the invention usingroutine experimentation.

[1135] As an example, for the cell lines tested (i.e. COS-7, CHO-S,HeLa, HT1080, BHK-21, and primary rat hippocampal and cortical neurons),transduction at an MOI of 50 followed by immediate transfection of thenucleic acid molecule encoding a fusion polypeptide of the inventiongenerally results in 50-80% suppression. This means that 50-80% of thepolypeptide expressed from the nucleic acid molecule encoding the fusionpolypeptide is expressed as the fusion polypeptide. Note that 100%suppression cannot be achieved at any MOI. Some untagged polypeptidewill always be expressed.

[1136] One of skill in the art will appreciate that the % suppression(i.e. suppression efficiency) achieved when cells are transduced at aparticular MOI (e.g. MOI=50) can vary and is dependent on a number offactors including: the amount of CAR expressed in the mammalian cell;the nature of the gene being expressed; the health of the cells at thetime of transduction; phenotypic changes to the cells resulting fromstop codon suppression.

[1137] Depending on the suppression efficiency and consequently, theamount of fusion polypeptide expressed, the % suppression achieved canbe optimized by varying the MOI using routine experimentation. It isimportant to note that while the % suppression achieved can be increasedby increasing the MOI, doing so may increase the likelihood ofphenotypic changes to the cells.

[1138] Expression of a suppressor tRNA in a host cell may result inphenotypic changes in the cell (e.g. toxicity) since ⅓ of the endogenousstop codons (i.e. all genes containing the stop codon recognized by thesuppressor tRNA) can be suppressed. This leads to potential addition ofextra amino acids to the C-termini of cellular proteins other than thefusion polypeptide of interest. In some embodiments, it may be desirableto optimize methods of the invention in order to minimize phenotypiceffects in a particular cell line of interest.

[1139] In embodiments of the invention where one or more suppressortRNAs are expressed from an adenovirus, the adenovirus may deliver atransient source of the suppressor tRNA to the target cell. When theadenovirus is replication-incompetent, it does not stably integrate intothe genome of the target cell, and will be diluted out gradually as celldivision occurs. This results in an overall decrease in suppressor tRNAexpression over time. In some embodiments, it may be desirable to detectfusion polypeptide of the invention from about 1 hour to about 5 days,from about 1 hour to about 4 days, from about 1 hour to about 3 day,from about 1 hour to about 2 day, from about 1 hour to about 1 day, fromabout 1 hour to about 20 hours, from about 1 hour to about 16 hours,from about 1 hour to about 12 hours, from about 1 hour to about 8 hours,from about 1 hour to about 4 hours, from about 1 hour to about 3 hours,from about 1 hour to about 2 hours, from about 8 hours to about 72hours, from about 8 hours to about 60 hours, from about 8 hours to about48 hours, from about 8 hours to about 36 hours, from about 8 hours toabout 24 hours from about 8 hours to about 20 hours, from about 8 hoursto about 16 hours, or from about 8 hours to about 12 hours aftertransduction of the cells of interest. Thus fusion polypeptide may bedetected at about 4 hours, about 8 hours, about 12 hours, about 16hours, about 20 hours, about 24 hours, about 28 hours, about 32 hours,about 36 hours, about 40 hours, about 44 hours, about 48 hours, about 52hours, about 56 hours, about 60 hours, about 64 hours, about 68 hours,about 72 hours, about 76 hours, about 80 hours, about 84 hours, about 88hours, or at about 92 hours after transduction of the cells.

[1140] Methods of the invention may be practiced using any number ofcells grown in any apparatus for that purpose known in the art (e.g.,tissue culture plates, tissue culture flasks, roller bottles,bioreactors, etc.) In some embodiments, tissue culture plates may beused (e.g., 6-well, 24-well, or 96-well plates). For high-throughputapplications, 96-well plates may be used. When cells are grown in tissueculture plates for use in the present invention, cells may be platedsuch that they are be 90% confluent at the time of transduction. As anexample, the cells may be transduced with an adenovirus expressing asuppressor tRNA at an MOI of 50 and transfected with a plasmid encodinga fusion polypeptide of the invention. As discussed above, any suitabletransfection protocol and/or reagents may be used. The amounts ofnucleic acid molecule encoding a fusion polypeptide of the invention andtransfection reagent may be adjusted to comport with the number of cellsto be transfected using techniques well known in the art.

[1141] In a non-limiting example, COS-7 cells may be plated at, forexample, 8×10⁴ COS-7 cells/per well of a 24-well plate and culturedovernight at 37° C. On the following day, the cells may be transduced(e.g., with an adenovirus expressing a suppressor tRNA), for example,using an MOI=50. It may be assumed that the number of cells has doubledduring overnight culture so that the number of cells is 2×8×10⁴=1.6×10⁵cells. If the titer of the viral stock is, for example, 1×10⁹ pfu/ml,the amount of viral stock to add to the cells may be calculated asfollows: 50 pfu/cell×1.6×10⁵ cells=8×10⁶ pfu/1×10⁹ (pfu/ml)=0.008 ml=8μl to add to each well.

[1142] In some embodiments, it may be desirable to include one or morecontrols in the methods of the invention. For examples, methods of theinvention may comprise transducing a host cell with a virus expressing asuppressor tRNA, transfecting the cells with a control nucleic acidmolecule (e.g., a nucleic acid molecule encoding a fusion polypeptidewith a reporter activity), and detecting a reporter activity. Forexample, pcDNA™6.2/GFP-GW/p64^(TAG) may be used as a positive controlfor transduction, transfection, and expression. In this plasmid, the p64protein (human c-myc) containing a TAG stop codon is cloned in framewith the cycle-3 GFP reporter gene (see (Chalfie, M., et al., Science263:802-805 (1994); Crameri, A., et al., Nature Biotechnol. 14:315-319(1996)). Including pcDNA™6.2/GFP-GW/p64^(TAG) plasmid when conductingtransduction and transfection methods of the invention allows detectinga reporter gene activity (e.g., assaying for cycle-3 GFP expressionusing fluorescence microscopy or c-myc expression using Western blotanalysis), and evaluating transfection and/or transduction conditions.

[1143] In one non-limiting example, methods of the invention may be usedto express a fusion polypeptide of the invention. Methods of theinvention may comprise seeding cells into a suitable tissue culturevessel at a suitable density (e.g., at a density such that the cellswill be approximately 90% confluent at the time of transduction).Optionally, cells may be incubated (e.g., at 37° C. overnight) afterseeding. When a 24-well tissue culture plate is used, cells may beseeded in 500 μl of complete medium. Methods of the invention maycomprise, on the day of transduction, removing the growth medium fromeach well of cells and replacing with fresh growth medium (for a 24-wellplate, 250 μl of medium may be used). Methods may further comprisecontacting the cells with a nucleic acid molecule encoding a suppressortRNA (e.g., transducing the cells with an adenovirus expressing asuppressor tRNA). When the nucleic acid molecule expressing a tRNA is avirus, any suitable MOI may be used (e.g., 50). Methods may furthercomprise returning the transduced cells to an incubator.

[1144] In embodiments where the host cell line is a stable cell linecomprising a nucleic acid molecule encoding a fusion polypeptide of theinvention, methods of the invention may comprise incubating cells (e.g.,for 5-6 hours at 37° C.) after introduction of a nucleic acid moleculeencoding a suppressor tRNA (e.g., after transduction with an adenovirusexpressing a suppressor tRNA). Typically, cells are incubated for atleast 5 hours as transduction efficiency will be decreased at shortertimes. Longer incubation time is possible (e.g. overnight), but will notincrease the transduction efficiency and may increase cell toxicity.Methods may further comprise removing the medium containing virus fromthe cells (e.g., after 5-6 hours), washing the cells (e.g., with 500 μlof fresh, complete growth medium in a 24-well plate), adding completegrowth medium (e.g., 500 μl of fresh, complete growth medium in a24-well plate) and incubating the cells under conditions sufficient toexpress a fusion polypeptide of the invention (e.g. at 37° C. in anincubator for a suitable period of time). Methods of the invention mayfurther comprise detecting the fusion polypeptide.

[1145] In some embodiments, a nucleic acid molecule encoding a fusionpolypeptide of the invention may be introduced into a host cell (e.g.,after the cell has been transduced as described above). For example,after transduction, a suitable amount of a nucleic acid moleculeencoding a fusion polypeptide of the invention may be mixed in asuitable medium. For example, for a well of a 24 well plate 500 ng ofplasmid DNA may be dissolved in 50 μl of Opti-MEM® I Reduced SerumMedium without serum and a suitable amount of a transfection reagent(e.g., a cationic lipid transfection reagent) may be mixed with asuitable amount of a medium (e.g., for a well of a 24 well plate, 1.5 μlof Lipofectamine™ 2000 may be mixed in 50 μl of Opti-MEMO® I ReducedSerum Medium). Both mixtures (i.e., DNA:medium and reagent:medium) maybe incubated, for example, for 5 minutes at room temperature. The twomixtures may be combined and incubated, for example, for 20 minutes atroom temperature to allow the formation of nucleic acid:transfectionreagent complexes (e.g., DNA-Lipofectamine™ 2000 Reagent complexes).Methods of the invention may comprise adding the complexes (e.g.,DNA-Lipofectamine™ 2000 Reagent complexes) directly to the growth mediumcontaining viruses used to transduce the host cells. Methods maycomprise incubating the cells (for example, for 5-6 hours at 37° C.).Typically, cells are incubated for at least 5 hours as transductionefficiency will be decreased at shorter times. Longer incubation time ispossible (e.g. overnight), but will not increase the transductionefficiency and may increase cell toxicity. Methods may further compriseremoving the medium containing virus from the cells (e.g., after 5-6hours), washing the cells (e.g., with 500 μl of fresh, complete growthmedium in a 24-well plate), adding complete growth medium (e.g., 500 μlof fresh, complete growth medium in a 24-well plate) and incubating thecells under conditions sufficient to express a fusion polypeptide of theinvention (e.g. at 37° C. in an incubator for a suitable period oftime). Methods of the invention may further comprise detecting thefusion polypeptide.

[1146] One skilled in the art can readily adjust the volumes of thevarious reagents described above to transduce cells in different tissueculture formats (e.g., vary the amounts of cells and medium used) inproportion to the difference in surface area of the tissue cultureplates used. For example, a 96-well plate may be used having a surfacearea per well of 0.3 cm², cells may be seeded in a volume of 100 μl andtransduced in a volume of 50 μl; a 6-well plate may be used having asurface are per well of 10 cm², cells may be seeded in a volume of 2 mland transduced in a volume of 1 ml.

[1147] As a non-limiting example, for methods of the invention usingCOS-7 cells, the following seeding densities and reagent quantities fortransduction and transfection may be used in different tissue cultureformats. Note that the suggested DNA quantities are for transfectionusing Lipofectamine™ 2000 Reagent. Condition 6-well 24-well 96-wellSeeding density 3 × 10⁵ cells  8 × 10⁴ cells  1 × 10⁴ cells MOI = 50 3 ×10⁷ virus  8 × 10⁶ virus  1 × 10⁶ virus Amount of plasmid DNA 2 μg 500ng 320 ng per well Amount of Lipofectamine ™ 6 μl  1.5 μl  1 μl 2000Reagent per well

[1148] In methods of the invention in which an adenovirus is used toexpress a suppressor tRNA in a host cell, one skilled in the art willrecognize that such expression is transient. Accordingly, expression ofa fusion polypeptide of the invention from a transiently transfectedplasmid generally peaks within 24-48 hours following transfection. Toobtain maximal levels of fusion polypeptide of the invention, cells maybe harvested and assayed for fusion polypeptide of the inventionexpression between 24 and 48 hours post-transfection. Since expressionconditions will vary depending on the nature of a particular fusionpolypeptide of the invention and its half-life, conditions describedabove may be optimized using routine experimentation to obtain maximallevels of fusion polypeptide expression.

[1149] Methods of the invention may comprise detecting a fusionpolypeptide of the invention. In some embodiments, detection may be byWestern blot and/or immunofluorescence. In methods of this type, anantibody that specifically binds to the polypeptide of interest portionof the fusion protein may be used. One skilled in the art willappreciate that this allows detection of fused and un-fused forms of thepolypeptide of interest. Alternatively, an antibody that specificallybind to the additional polypeptide sequences of the fusion polypeptideof the invention allows detection of only the fusion polypeptide and notthe polypeptide of interest lacking the additional polypeptidesequences.

[1150] In some embodiments, additional polypeptide sequences in a fusionpolypeptide of the invention may be a fluorescent polypeptide (e.g., thegreen fluorescent protein (GFP)). In embodiments of this type, it may bedesirable to detect the fluorescence of the fluorescent polypeptide. Ina specific embodiment, methods of the invention may comprise detectingGFP. GFP may be detected, for example, in vivo using fluorescencemicroscopy. An example of a fusion polypeptide of the invention is theGFP-tagged p64^(TAG) fusion protein expressed from the plasmidpcDNA™6.2/GFP-GW/p64^(TAG). Since the GFP-tagged p64^(TAG) protein isexpressed from the strong CMV promoter, fusion protein is generallydetectable within 24 hours after transfection.

[1151] To detect fluorescent cells, suitable filter sets to optimizedetection may be employed. The primary excitation peak of cycle-3 GFP isat 395 nm. There is a secondary excitation peak at 478 nm. Excitation ateither of these wavelengths yields a fluorescent emission peak with amaximum at 507 nm. Note that the quantum yield can vary as much as 5- to10-fold depending on the wavelength of light that is used to excite theGFP fluorophore.

[1152] Use of the best filter set will insure that the optimal regionsof the cycle-3 GFP spectra are excited and passed (emitted). A filterset designed to detect fluorescence from wild-type GFP (e.g. OmegaOptical XF76 Filter) may be used. Alternatively, FITC filter sets may beused to detect cycle-3 GFP fluorescence. One skilled in the art willappreciate that these filter sets are not optimal and fluorescent signalmay be weaker. For example, a typical FITC filter set excites cycle-3GFP with light from 460 to 490 nm, covering the secondary excitationpeak. The filter set passes light from 515 to 550 nm, allowing detectionof most but not all of the cycle-3 GFP fluorescence.

[1153] Most tissue culture media fluoresce because of the presence ofriboflavin (see, Zylka, M. J., and Schnapp, B. J. (1996) BioTechniques21, 220-226) and may interfere with detection of cycle-3 GFPfluorescence. To alleviate this problem, methods of the invention maycomprise removing the growth medium and replacing the growth medium withPhosphate-Buffered Saline (PBS; Invitrogen Corporation, Carlsbad,Calif., Catalog no. 10010-023) before assaying for GFP fluorescence. Ifcells are being cultured further after assaying, methods of theinvention may comprise removing the PBS and replacing with fresh growthmedium prior to re-incubation.

[1154] In some embodiments, methods of the invention may comprisedetecting a fusion polypeptide of the invention comprising a polypeptidesequence of the GFP by Western blotting. For example, GFP-taggedp64^(TAG) fusion polypeptide can be detected by Western analysis usingthe following antibodies available from Invitrogen Corporation,Carlsbad, Calif.: to detect both untagged and GFP-tagged p64^(TAG)protein, an antibody that specifically binds to the p64 portion of thefusion polypeptide (i.e., one of the Anti-myc Antibodies) can be used;to detect GFP-tagged p64^(TAG) protein only, an antibody thatspecifically binds to the GFP portion of the fusion polypeptide (e.g.,the GFP Antiserum) may be used.

[1155] In methods of the invention that comprise detecting a fusionpolypeptide of the invention by Western blotting, a lysate of host cellsexpressing the fusion polypeptide may be prepared. For example, a celllysate to assay for native or GFP-tagged p64 protein may be prepared.One skilled in the art will appreciate that procedures using NP-40 lysisare not effective in releasing p64 protein. Since p64 is localized inthe nucleoli, harsher lysis procedures using RIPA or SDS-PAGE samplebuffer may be used to adequately solubilize p64 in total cell lysates.Methods of preparing a cell lysate to assay for p64 protein, maycomprise washing cell monolayers (e.g., washing once withPhosphate-Buffered Saline Invitrogen Corporation, Carlsbad, Calif., PBS,Catalog no. 10010-023); adding a suitable lysis buffer (e.g., 1×SDS-PAGESample Buffer) to each well containing cells (e.g., for a 24-well plate,100 μl of 1×SDS-PAGE Sample Buffer per well may be used); looseninglysed cells from the plate (e.g., e a pipette tip can be used to loosenlysed cells from plate); and transferring the cells to a centrifuge tube(e.g., for cells from one well of a 24-well late a 1.5 mlmicrocentrifuge tube). Lysates will be viscous.

[1156] Methods of preparing a lysate may further include heating samplesat 70° C. for 10 minutes. Optionally, methods may include mixing (e.g.using a vortex mixer) one or more times and briefly centrifuging thesample. A lysate prepared by methods of the invention may be furtherprocessed or analyzed using techniques well known in the art. Forexample, an aliquot of the lysate (e.g., 5 μl of cell lysate) may beloaded onto an SDS-PAGE gel and electrophoresed. The GFP-taggedp64^(TAG) protein has a molecular weight of approximately 77.2 kDa.

[1157] One example of a suitable lysis buffer is 1× SDS-PAGE SampleBuffer, which may be prepared by combining the following reagents in theamounts indicated: 0.5 M Tris-HCl, pH 6.8 (2.5 ml); Glycerol (100%) (2ml); β-mercaptoethanol (0.4 ml); Bromophenol Blue (0.02 g); SDS (0.4 g);and sterile water to a final volume of 20 ml. Aliquots of the buffer maybe frozen at −20° C. until needed.

[1158] In one specific embodiment, the following ORFs were amplified tocontain a TAG stop codon, and cloned into the pENTR/D-TOPO® Gateway®vector to generate entry clones. The entry clones were then transferredinto the pcDNA™6.2/GFP-DEST vector using the Gateway® LR recombinationreaction to create expression clones: 1) human CGI-130 (GenBankAccession # BC003357), which localizes to the cytoplasm; 2) humannuclear splicing factor(GenBank Accession # BC000997), which localizesin the nucleus; and 3) human c-myc (GenBank Accession # BC000141), whichlocalizes with the nucleoli. COS-7 cells were transduced with the anadenovirus expressing suppressor tRNA molecules (i.e., Tag-On-Demand™Suppressor Supernatant, Invitrogen Corporation, Carlsbad, Calif.) at anMOI of 50 followed by transfection with the pcDNA™6.2/GFP-DESTexpression constructs using the procedure described above. Twenty-fourhours post-transfection, GFP fluorescence was assayed using fluorescencemicroscopy. Fluorescent micrographs for each expression construct areshown in FIG. 64. For all three proteins above, methods of the inventionresult in expression of detectable levels of GFP-tagged recombinantprotein as measured by GFP fluorescence by 24 hours post-transfection.Also, the GFP-tagged recombinant protein is correctly localized to theappropriate cellular organelle. The expression construct containing ORF3(BC000141) is the same construct as the controlpcDNA™6.2/GFP-GW/p64^(TAG) plasmid described above.

[1159] In some instances, cell toxicity may be observed whentransduction and transfection are performed sequentially with a 5-6 hourincubation period after transduction. Although suppression may beclearly observed under these circumstances, the cells may appearunhealthy and may detach from the plate. This phenomenon is not due toeither virus alone or transfection alone.

[1160] It has been demonstrated that adenovirus transduction performedsimultaneously with plasmid transfection results in reduced toxicity andincreased plasmid-derived gene expression (see Cotten et al., Proc NatlAcad Sci USA 89(13):6094-8, (1992); Curiel et al., Proc Natl Acad SciUSA 88(19):8850-4, (1991); Guy et al., Mol Biotechnol. 3(3):237-48,(1995); Honda et al., J Virol Methods 58(1-2):41-51, (1996); Merwin etal., J Immunol Methods 186(2):257-66, (1995); Zatloukal et al., VerhDtsch Ges Pathol. 78:171-6, (1994)).

[1161] A series of experiments were performed to directly compare themethod of sequential transduction-transfection with a simultaneoustransduction/transfection method. In addition to being easier toperform, the simultaneous method resulted in cells that were clearlyhealthier (normal morphologies and proper adherence) with no evidence oftoxicity (FIG. 66, right panels) as compared to the sequential method(left panels). As an added benefit, transfection efficiencies werehigher making detection of fluorescent cells easier. In FIG. 66, 8×10⁴COS-7 cells were plated in 24-well format and transfected/transduced asfollows: Sequential Method (left panels): Cells were transduced with anadenovirus expressing a suppressor tRNA molecule (Ad-tRNA^(TAG)) at anMOI of 50 for 5 hours, media was replaced and cells were grownovernight. The following morning, cells were transfected with 0.5 μgpcDNA6.2/GFP-GW/p64^(TAG) using 1.5 μl Lipofectamine 2000 for 6 hours,media was replaced and cells grown overnight. GFP fluorescence andbrightfield microscope photos were taken the following day. SimultaneousMethod (right panels): Cells were transfected/transduced simultaneously.Adenovirus expressing a suppressor tRNA (Ad-tRNA^(TAG)) at an MOI of 50was applied to cells and pre-formed DNA:Lipid complexes (0.5 μg DNA+1.5μl Lipofectamine 2000) were added directly to the virus and cells for 5hours. Media was replaced and GFP fluorescence and brightfieldmicroscope photos were taken the following day.

[1162] A variety of lipid/DNA ratios were also evaluated using thesimultaneous transduction/transfection method (FIG. 67). All lipid/DNAratios tested resulted in healthy, normal looking cells. Westernblotting revealed that all ratios tested gave stop suppression greaterthan 50%, even at MOI 25, with suppression levels ranging from 63% to87% when simultaneous transduction/transfection was used (FIG. 67, upperpanels). In FIG. 67, 8×10⁴ COS-7 cells were plated in 24-well format andtransfected/transduced as described for Sequential and Simultaneousmethods above. Various Lipid/DNA ratios were tested, as indicated. 24hours post transduction/transfection, 5 μl of each total cell lysate wasanalyzed on 4-12% NuPage gel, MOPS running buffer, transferred to PVDFmembrane and Western blot probed with anti-myc antibody. Percentsuppression was determined by densitometry

[1163] Gene expression levels were noticeably higher with thesimultaneous method and there was no MOI-dependent shut-down of geneexpression (i.e. no MOI-dependent toxicity) which was visible with thesequential method (FIG. 67, compare upper western blot panels with lowerpanels).

[1164] Thus, methods of producing a fusion polypeptide according to theinvention may comprise transducing a host cell with an adenovirusexpressing a suppressor tRNA and introducing a nucleic acid moleculeencoding a fusion polypeptide into the host cell wherein the host cellis contacted with the adenovirus and the nucleic acid molecule at thesame time. Such a method may comprise seeding host cells, transducinghost cells with an adenovirus and contacting host cells with one or morecomplexes comprising one or more nucleic acid molecules and one or moretransfection reagents. Adenovirus may be used at any suitable MOI asdiscussed above (for example, about 50). Methods may comprise incubatingcells in the presence of adenovirus and complexes for from about 10minutes to about 48 hours, from about 10 minutes to about 36 hours, fromabout 10 minutes to about 24 hours, from about 10 minutes to about 20hours, from about 10 minutes to about 16 hours, from about 10 minutes toabout 12 hours, from about 10 minutes to about 8 hours from about 10minutes to about 7 hours, from about 10 minutes to about 6 hours, fromabout 10 minutes to about 5 hours, from about 10 minutes to about 4hours, from about 10 minutes to about 3 hours, from about 10 minutes toabout 2 hours, from about 10 minutes to about 1 hour, from about 10minutes to about 45 minutes, from about 10 minutes to about 30 minutes,from about 1 hour to about 48 hours, from about 1 hour to about 36hours, from about 1 hour to about 24 hours, from about 1 hour to about20 hours, from about 1 hour to about 16 hours, from about 1 hour toabout 12 hours, from about 1 hour to about 8 hours, from about 1 hour toabout 7 hours, from about 1 hour to about 6 hours, from about 1 hour toabout 5 hours, from about 1 hour to about 4 hours, from about 1 hour toabout 3 hours, from about 1 hour to about 2 hours, from about 2 hours toabout 48 hours, from about 3 hours to about 48 hours, from about 4 hoursto about 48 hours, from about 5 to about 48 hours, from about 6 hours toabout 48 hours, from about 7 hours to about 48 hours, from about 8 hoursto about 48 hours, from about 9 hours to about 48 hours, or from about10 hours to about 48 hours. Thus, cells may be incubated in the presenceof virus and nucleic acid molecule about 1 hour, about 2 hours, about 3hours about 4 hours, about 5 hours, about 6 hours, about 7 hours, about8 hours, about 9 hours, about 10 hours about 12 hours, about 16 hours,about 20 hours, about 24 hours, about 36 hours or about 48 hours.

[1165] Methods may further comprise removing the solution comprisingvirus and complexes; contacting the cells with a suitable medium (e.g.,a complete medium); and incubating the cells under conditions sufficientto produce a fusion polypeptide of the invention. Methods may furthercomprise detecting the fusion polypeptide using any technique known tothose skilled in the art (e.g., fluorescence microscopy, westernblotting, etc). In some instances, toxicity may be observed at latertime points and may be cell type specific. Cells should be closelymonitored for evidence of toxicity if incubations are carried out forextended periods of time (e.g., longer than about 24-48 hours).

[1166] Suitable amounts of cells, media, virus, DNA, and transfectionreagent (Lipofectamine 2000=LF2K) for various size tissue culture traysare as follows 6-well 24-well 96-well COS cells seeded per well 3 × 10⁵cells  8 × 10⁴ cells  1 × 10⁴ media/well for culturing 2 ml 500 μl 100μl MOI 50 3.0 × 10⁷ virus  8 × 10⁶ virus  1 × 10⁶ virus MOI 25 1.5 × 10⁷virus  4 × 10⁶ virus  5 × 10⁵ virus media/well during tdx/tfx 1 ml 250μl  50 μl Transfect DNA/well 2 μg 500 ng 320 ng LF2K/well 6 μl  1.5 μl 1 μl

[1167] Methods of the invention may comprise one or more incubationsteps that may be performed in complete medium as described herein,unless otherwise indicated. Cell numbers are for COS-7 cells. Other celltypes may require different cell numbers. Cells should be ˜90% confluenton the day of virus transduction. Assume that the number of cells doublefrom the time of seeding to the time of transduction for the purpose ofcalculating MOI.

[1168] An example of a complete media is DMEM (high glucose)supplemented with FBS to a final concentration of 10%, L-glutamine to afinal concentration of 4 mM, and MEM non-essential amino acids to afinal concentration of 0.1 mM. These reagents are commercially availablefrom, for example, Invitrogen Corporation, Carlsbad, Calif. (DMEM (highglucose) catalog no. 11960-044, FBS catalog no. 16000-044, L-glutamine(200 mM) catalog no. 25030-081, MEM non-essential amino acids (10mM=100×) catalog no. 11140-050). Media should not be warmed in a waterbath. Media should be allowed to come to room temperature in the dark.

[1169] As discussed above, in some embodiments, methods of the inventionmay comprise making a cell lysate. One suitable method for making alysate entails removing media from the wells, adding a suitable lysisbuffer (e.g., for a 24-well plate, 100 μl of 2× NuPAGE® LDS SampleBuffer (4×NuPAGE® LDS sample buffer is available from InvitrogenCorporation, Carlsbad, Calif., catalog no. NP0007 and can be dilutedwith water)) with {fraction (1/50)}^(th) volume of β-mercaptoethanol toeach well; loosening the cells from the plate (e.g., using a pipette tipin a swirling motion to loosen lysed cells from plate); and transferringto a centrifuge tube (e.g., 1.5 ml eppendorf tube). Typically, lysatesmay be viscous. If it is too viscous, more 2×NuPage LDS Sample Bufferwith β-mercaptoethanol can be added up to a total of 200 μl. Samplesshould be stored at −80° C. until conclusive western blotting has beencompleted.

[1170] Methods may further entail heating samples (e.g., at 70° C. for10 minutes); mixing samples one or more times (e.g., vortexing andcentrifugation throughout); loading an aliquot of the sample on anSDS-PAGE gel (e.g., loading 5 μl (per 100 μl harvested) on a 4-12%NuPage Bis-Tris gel, Invitrogen Corporation, Carlsbad, Calif., catalogno. NP0322BOX). Each gel may contain one or more controls molecularweight markers (e.g., 5 μl of Magic Mark, Invitrogen Corporation,Carlsbad, Calif., catalog no. LC5600, 10 μl of See Blue Plus 2,Invitrogen Corporation, Carlsbad, Calif. LC5925), and Western blotcontrols (e.g., 10 μl of Positope, Invitrogen Corporation, Carlsbad,Calif. R90050) heated at 70° C. Electrophoresis-may be performed using,for example, 1×NuPage MOPS SDS Sample Running Buffer (InvitrogenCorporation, Carlsbad, Calif., catalog no. NP0001). Add 500 μl of NuPageAntioxidant (Invitrogen Corporation, Carlsbad, Calif., catalog no.NP0005) to the sample running buffer in the “inner core.”Electrophoresis may be performed for a suitable period of time undersuitable conditions (e.g., for approximately 50 minutes at 200 volts).

[1171] For Western blotting of gel, make up a suitable transfer buffer(e.g., 1×NuPage Transfer Buffer with 20% methanol, InvitrogenCorporation, Carlsbad, Calif. catalog no. NP0006). Add 1 ml ofAntioxidant to 1 liter of 1×NuPage Transfer Buffer. Wet PVDF membranes(Invitrogen Corporation, Carlsbad, Calif. catalog no. LC2002) inmethanol, rinse with H₂O, and then equilibrate in Transfer Buffer.Transfer to PVDF membrane for 90 minutes at 30 volts. Follow allprocedures and recommendations in NuPage Bis-Tris gel package insert(Invitrogen Corporation, Carlsbad, Calif.).

[1172] Following transfer, wash membrane 2× with 20 ml of H₂O. Blockmembrane using a suitable blocking solution (e.g., that provided in theanti-mouse Western Breeze Chemiluminescent Kit, Invitrogen Corporation,Carlsbad, Calif., catalog no. WB7104). Blocking can be done for 30minutes up to overnight. Dilute suitable antibody in an appropriatebuffer (e.g., for detecting myc protein, anti-myc antibody (InvitrogenCorporation, Carlsbad, Calif., catalog nos. R95025, R95225, or 95325)can be diluted 1:5000 in PVDF primary antibody diluent. Incubateantibody solution with membrane, wash, and detect bound antibody usingstandard techniques suitable for the antibodies used (e.g.,chemiluminescent detection, fluorogenic detection, radiolabel detection,etc.). Such techniques are well known to those skilled in the art.

[1173] When using myc protein that becomes tagged with GFP uponsuppression of the stop codon between the coding region of the twoproteins (e.g., as expressed from pcDNA6.2/GFP-GW/p64^(TAG)), un-taggedmyc (no virus control) should band around Magic Marks 55 kDa and myctagged with GFP should band around Magic Marks 80 kDa. Densitometry canbe done to determine % shift from untagged myc to GFP tagged myc (i.e.,percent suppression).

[1174] Other suitable lysis techniques may be used. For example, harvestcells from 24 well plate with 100 μl of 1×Tris-Glycine Sample Buffer(Invitrogen Corporation, Carlsbad, Calif., catalog no. LC2676)containing {fraction (1/50)}^(th) volume of β-mercaptoethanol to eachwell. Use a pipette tip in a swirling motion to loosen lysed cells fromplate and transfer to a 1.5 ml eppendorf tube. Lysates will be viscous,this is normal. If it is too viscous, more 1×Tris Glycine Sample Bufferwith β-mercaptoethanol can be added up to a total of 200 μl. Samples canbe stored at 4° C. Heat samples at 100° C. for 10 minutes (withvortexing and centrifugation throughout) prior to loading 5 μl (per 100μl harvested) on a 4-20% Tris Glycine gel (Invitrogen Corporation,Carlsbad, Calif., EC60252BOX). Western blot analysis may be performed asabove or using other suitable techniques know to those skilled in theart.

[1175] Another suitable lysis technique is as follows. Harvest cellsfrom 24 well plate with 100 μl of 1×RIPA lysis buffer containingComplete Protease Inhibitor Cocktail (Roche, catalog no. 1 697 498) 50×in H₂O) & Pepstatin (Roche, catalog no. 253 286) 1000× in EtOH). Usepipette tip in a swirling motion to loosen lysed cells from plate andtransfer to a 1.5 ml eppendorf tube. Lysates will be viscous. If it istoo viscous, more RIPA lysis buffer can be added up to 150 μl total.These lysates can be analyzed as above or using other techniques know tothose skilled in the art. Bradford Protein assay can be conducted withthis lysis buffer to quantitate total amount of protein loaded. Storesamples at −80° C. until ready to use. Thaw at room temperature, andthen keep on ice.

[1176] One suitable protocol for conducting the Bradford protein assayis as follows:

[1177] In a 96 well U-bottom flexible polyvinyl chloride plate (FalconCat. No. 35-3911)

[1178] Perform a 1:10 dilution of cell lysates (e.g., prepared asdescribed above) directly in the wells (9 μL of H₂O and 1 μL of lysate).

[1179] Load 10 μL of BSA standard curve to the 96 well plate (1000 μg/mlserial diluted 1:2 down to 15.625 μg/ml)

[1180] Add 190 μL Bradford reagent to 10 μL of diluted lysates andstandard curve (1:5 dilution of BioRad Protein Assay Solution, Bio-RadCorporation, Hercules, Calif., catalog no 500-002, 1 ml Solution and 4ml H₂O)

[1181] Read at endpoint wavelength 595 on plate reader and displayReduced numbers.

[1182] Use 4 parameter fit for Graph.

[1183] The methods described above may scaled up or down as appropriatefor the number of cells to be used. In some embodiments, particularlythose involving high-throughput applications, it may be desirable toanalyze a large number of samples in a 96-well format. The protocol for96-well late applications is the same as the 24 well format describedpreviously with the following modifications.

[1184] Seed COS-7 cells at 1×10⁴ cells/well in 100 μl/ well in a 96-wellplate.

[1185] Assume doubling of cells in 24 hour period to 2×10⁴ cells/well.

[1186] Transduction & Transfection are conducted in 50 μl/well volumes(100 μl total- 50 culture media, 50 complexes) for 5 hours. Complexesmay be formed using 25 μl medium (e.g., 1×OPTIMEM) and 1 μl transfectionreagent (e.g., Lipofectamine 2000) and 25 μl medium and 320 ng DNAincubated separately for 5 minutes at room temperature and then combinedand incubated for 20 minutes at room temperature.

[1187] Cells may be harvested with 30-60 μl of Sample Buffer or 30-50 μlLysis Buffer, depending upon viscosity.

[1188] Load 10 μl (per 30 μl harvested) of samples harvested with SampleBuffer on gel.

[1189] The most likely sources of low suppression efficiency includepoor quality of cell stock at time of plating experiment (i.e., cellsvery confluent, media not pink) and the use of old media. Media shouldbe freshly prepared for use in transduction/transfection.

[1190] In another specific example of methods of the invention, COS-7cells were transduced with an adenovirus expressing suppressor tRNAmolecules (i.e., the Tag-On-Demand™ Suppressor Supernatant) at variousMOIs following the procedures described above and simultaneouslytransfected with the pcDNA™6.2/GFP-GW/p64^(TAG) plasmid usingLipofectamine 2000 Reagent and the procedure described above.Twenty-four hours post-transfection, cell lysates were prepared andanalyzed by Western blot using the Anti-myc Antibody and theWesternBreezee® Chemiluminescent Anti-Mouse Kit (Catalog no. WB7104) todetect native and GFP-tagged p64^(TAG) (c-myc) protein. The results areshown in FIG. 68. In FIG. 68, Lane 1 contains MagicMark™ MW Standard,lane 2 contains untransfected COS-7 cells, lane 3 contains cellstransduced at an MOI=0, lane 4 contains cells transduced at an MOI=50,lane 5 contains cells transduced at an MOI=100, lane 6 contains cellstransduced at an MOI=200. GFP-tagged c-myc protein is produced anddetectable by Western blot within 24 hours post-transfection. The %suppression achieved is >80% when transducing cells at an MOI≧50. Inthis experiment, increasing the MOI has little effect on the suppressionefficiency. Maximal levels of GFP-tagged c-myc protein are producedusing an MOI=50.

[1191] In another working example of methods of the invention, 96 ofInvitrogen's Ultimate™ Human ORF Clones encoding 96 different kinaseswere transferred into the pcDNA™6.2/V5-DEST vector using the Gateway® LRrecombination reaction to generate expression clones. The expressionconstructs were purified, and the plasmid DNA (ranging from 20 ng to 300ng) was transfected using Lipofectamine™ 2000 Reagent into COS-7 cells(plated in 96-well format) that had been transduced with theTag-On-Demand™ Suppressor Supernatant at an MOI of 50 following theprocedure described above. Forty-eight hours post-transfection, celllysates were prepared and analyzed by Western blot using the Anti-V5Antibody (Invitrogen, Catalog no. R961-25) and the WesternBreeze®Chemiluminescent Anti-Mouse Kit (Catalog no. WB7104) to detect V5-taggedfusion polypeptide. Using this antibody, native polypeptide is notdetected. V5-tagged fusion polypeptide is produced and detectable byWestern blot within 48 hours post-transfection. The levels of V5-taggedfusion polypeptide produced vary widely from gene to gene. This isexpected since transfection and expression conditions are not optimizedfor each gene and can vary depending on the nature of the gene ofinterest. In this working example, the amount of plasmid DNA transfectedand the amount of cell lysate loaded on the polyacrylamide gel were notquantitated for each sample (i.e. transfection and expression conditionswere not optimized). In addition, antibodies to each of the 96 differentkinase proteins do not exist. This example demonstrates the utility ofmethods of the invention to quickly screen and analyze the expression oflarge numbers of recombinant proteins for which antibodies do notcurrently exist.

[1192] As discussed above, methods of the invention may be optimizedusing routine experimentation in order to produce a desired quantity offusion polypeptide of the invention. A variety of factors may beconsidered when optimizing experimental conditions. For example, in someinitial experiments, low expression of the desired fusion polypeptidemay be observed. This may be due to any one or more or a number ofreason such as 1) low suppression efficiency; 2) phenotypic effectsobserved; 3) poor transfection efficiency; and 4) improper timing of theassay (i.e., assayed too early or too late).

[1193] Low suppression efficiency may result in a reduced production ofa desired fusion polypeptide and may be observed when the host cellsused were not healthy and/or were not plated at the correct density. Oneskilled in the art can optimize this factor by ensuring that cells arehealthy and >95% viable before plating and are plated at the properdensity. Low suppression efficiency may be observed when the media usedwas not fresh. This factor can be optimized by preparing fresh media foruse in the practice of the present invention. Low suppression efficiencymay be observed if the host cells are transduced with too little virus(i.e. low MOI). One skilled in the at can optimize transduction bytesting varying MOIs starting at about 50. Low suppression efficiencymay be observed when host cells express low levels of CAR. One skilledin the art can optimize this factor by using a cell line that expressessuitable levels of CAR (e.g. COS-7, CHO, HeLa). Low suppressionefficiency may be observed if host cells are not transduced for anoptimum length of time. One skilled in the art can optimize this factorby transducing for various periods of time, for example, about 5-6hours.

[1194] Phenotypic effects on host cells caused by methods of theinvention may result in reduced production of a desired fusionpolypeptide of the invention. Factors that may be optimized to mitigatephenotypic effects include the length of incubation after transductionand transfection. One skilled in the art can optimize this factor byassaying for fusion polypeptide at various times after transduction andtransfection (e.g., 24-48 hours). Phenotypic effects may be observed ifhost cells used are sensitive to transduction and transfectionprocedure. One skilled in the art can optimize this factor by performingmethods of the invention in a different host cell line and/or by makinga stable cell line containing the nucleic acid molecule encoding thefusion polypeptide and subsequently introducing a nucleic acid moleculeencoding a suppressor tRNA (e.g., transducing with a virus expressing asuppressor tRNA).

[1195] Poor transfection efficiency may result in a reduced productionof a desired fusion polypeptide of the invention. One skilled in the artcan readily optimize this factor by testing various transfection reagentto identify one that provides a high transfection efficiency for thecell line being used.

[1196] Reduced production of a fusion polypeptide of the invention maybe observed when fusion polypeptide expression is assayed at asub-optimal time (i.e., too early or too late). One skilled in the artcan optimize this factor by assaying at various times to determine whenoptimum expression is observed (e.g., by conducting a time course ofexpression).

Example 17

[1197] In some embodiments, the invention provides nucleic acidmolecules comprising all or a portion of a viral genome that comprisetranscriptional regulatory sequences (e.g., promoters, repressors,etc.). In one specific embodiment, the invention provides nucleic acidmolecules comprising all or a portion of a viral genome (e.g., aretroviral genome) that comprise a repressor sequence. A repressorsequence may inhibit or prevent transcription of a nucleotide sequenceto which it is operably linked.

[1198] A repressor sequence may bind or may be bound by one or moremolecules (e.g., peptides, small molecules, etc.). In one embodiment, arepressor sequence may bind a protein (e.g., a repressor protein). Oneexample of a repressor to which binds a repressor protein is thetetracycline operator to which binds the tetracycline repressor protein.In the absence of tetracycline, the repressor protein binds to thetetracycline operator and prevents or inhibits transcription of anucleotide sequence to which it is operably linked. In the presence oftetracycline, the repressor protein binds tetracycline and no longerbinds to the repressor sequence.

[1199] In some embodiments, a repressor sequence and a promoter sequencemay be operably linked to a sequence of interest. In embodiments of thistype, the repressor sequence may prevent transcription of the sequenceof interest from the promoter under some conditions (e.g., when arepressor protein is bound to the repressor sequence) and not underother conditions (e.g., in the absence of repressor protein or underconditions in which the repressor protein is not bound to the repressorsequence).

[1200] In one embodiment of the invention, a nucleic acid moleculecomprising all or a portion of a lentiviral genome may also comprise arepressor sequence (e.g., the tetracycline operator) and/or may comprisea nucleic acid sequence encoding a polypeptide that binds to a repressor(e.g., the tetracycline repressor protein). Embodiments of this type maybe used to construct host cells and/or host cell lines comprising anucleic acid sequence of interest operably linked to a repressorsequence. Optionally, such host cells and/or host cell lines maycomprise a nucleic acid sequence encoding a polypeptide that binds tothe repressor sequence. In a particular embodiment, the presentinvention encompasses host cells and/or host cell lines in which asequence of interest is operably linked to a tetracycline repressorsequence and a promoter sequence and further comprise a nucleic acidsequence encoding the tetracycline repressor protein. Such host celllines provide the ability to regulate the transcription of the sequenceof interest, i.e., in the absence of tetracycline, the sequence ofinterest is not transcribed or is transcribed at an insignificant levelwhile in the presence of tetracycline the sequence of interest istranscribed at a much higher level (i.e., transcription is induced bytetracycline).

[1201] Host cells and/or host cell lines according to the invention maybe any type of cell (e.g., dividing or non-dividing cells) and may beisolated cells or may be within a larger organism. Methods of theinvention allow controlled gene expression in tissue culture cells andwhole organisms.

[1202] In some embodiments, the present invention provides a method ofmaking a cell expressing a repressor protein and cells made by suchmethods. Methods may comprise introducing into a cell a nucleic acidmolecule comprising all or a portion of a viral genome and encoding arepressor protein. Such methods may also comprise selecting for a cellstably expressing the repressor.

[1203] In some embodiments, the present invention comprises methods ofexpressing a sequence of interest comprising introducing into a hostcell expressing a repressor protein, one or more nucleic acid moleculescomprising a sequence of interest operably linked to a repressor and apromoter. In some embodiments, a nucleic acid molecule comprising asequence of interest operably linked to a repressor and a promoter maycomprise all or a portion of a viral genome (e.g., a lentiviral genome).Methods may further comprise incubating the cell under conditions inwhich the repressor protein does not bind to the repressor sequence.Such conditions may include incubation in the presence of a moleculethat prevents the repressor protein from binding to the repressorsequence. For example, when the repressor sequence is the tetracyclineoperator and the repressor protein is TetR, such conditions may compriseincubating the cell in the presence of tetracycline.

[1204] In one particular embodiment, the present invention provides twonucleic acid molecules (e.g., plasmids, viral vectors etc.) that may beused in the practice of the invention. A first nucleic acid moleculecomprises a repressor sequence and a promoter and may comprise asequence of interest operably linked to the repressor and promoter. Afirst nucleic acid molecule may also comprise one or more recognitionsequences (e.g., recombination sites, topoisomerase sites, restrictionenzyme sites, etc.). One non-limiting example of a first nucleic acidmolecule is the plasmid, pLenti4/TO/V5-DEST, which contains two copiesof the tetracycline operator sequence (TO) within the CMV promoter(CMVTetO₂). A map of this vector is provided as FIG. 70A and thenucleotide sequence is provided in Table 31. This plasmid also containstwo recombination sites that do not recombine with each other. Asequence of interest may be operably linked to the promoter andrepressor using any technique known in the art. In one embodiment, asequence of interest may be operably linked to the promoter andrepressor by conducting a recombination reaction between a sequence ofinterest flanked by recombination sites and the nucleic acid molecule ofthe invention. For example, pLenti4/TO/V5-DEST (FIG. 70A) can be reactedwith a sequence of interest flanked by attR1 and attR2 sites to operablylink the sequence of interest to the CMV promoter and tetracyclineoperator in a LR-recombination reaction. The reaction places thesequence of interest downstream of CMVTetO₂ for regulated expression inthe presence of the tetracycline repressor protein.

[1205] A second nucleic acid molecule of the invention may express oneor more proteins that interact with repressor sequences. Onenon-limiting example of a repressor protein is the tetracyclinerepressor protein (TetR). One example of a suitable second nucleic acidmolecule is the repressor plasmid pLenti6/TR, which expresses TetR. Amap of this vector is provided as FIG. 69 and the nucleotide sequence isprovided as Table 32. TetR binds the tetracycline operator sites inCMVTetO₂ promoter on the expression vector and blocks transcription fromthe promoter in the absence of inducer. When tetracycline inducer bindsTetR, however, the latter dissociates from the promoter andtranscription proceeds.

[1206] Methods of the of the invention may be use to regulate theexpression of a sequence of interest in transformed dividing cells andin difficult-to-transfect growth-arrested primary cells. Methods of theinvention may be used for transient or stable gene regulation. Inductionof expression may be from about 2-fold to about 100-fold, from about5-fold to about 100-fold, from about 10-fold to about 100-fold, fromabout 25-fold to about 100-fold, from about 50-fold to about 100, fromabout 75-fold to about 100-fold, from about 5-fold to about 5-fold toabout 70-fold, from about 10-fold to about 70-fold, from about 25-foldto about 70-fold, from about 50-fold to about 70-fold, or from about60-fold to 70-fold. Thus, gene expression may be induced about 5-fold,about 10-fold, about 20-fold, about 30-fold, about 40-fold, about50-fold, about 60-fold, about 70-fold, about 80-fold, about 90-fold, orabout 100-fold.

[1207] In some embodiments of the invention, the present invention maycomprise a viral stock that may be used to transduce host cells. Stocksmaybe at any suitable concentration of virus. For example, pLenti6/TRmay be used to create a viral stock at about 1×10⁵ cfu/ml or greater,which may be used to stably transduce TetR into target cells inblasticidin-containing media. Cells transduced in this fashion willtypically express TetR protein at a level detectable by Western blot.

[1208] In another aspect of the invention, the present inventionprovides nucleic acid molecules comprising a promoter sequence and arepressor sequence to which a sequence of interest may be operablylinked. Such nucleic acid molecules may be used to create a viral stock.For example, recombinational cloning of the lacZ gene intopLenti4/TO/V5-DEST and packaging the resulting pLenti4/TO/V5-GW/lacZvector may be used to produce a viral stock at 1×10⁵ cfu/ml or greater.Such a viral stock may be used to transduce a host cell and express asequence of interest (e.g., the lacZ sequence).

[1209] In some embodiments, nucleic acid molecules comprising a promoterand repressor sequence operably linked to a sequence of interest andnucleic acid molecules comprising a sequence encoding a polypeptide thatbinds to the repressor sequence may be introduced into a host cell. Inmethods of this type, nucleic acid molecules may be introducedsimultaneously or sequentially. Typically, once both types of nucleicacid molecule have been introduced into a host cell, expression of thesequence of interest will be inducible. For example, transientco-transduction of Lenti6/TR and Lenti4/TO/V5-GW/LacZ may show at least20-fold induction in HT1080 cells. In some embodiments, after both typesof nucleic acid molecule are introduced into a host cell, a stable cellline may be produced, for example, by selecting for cells expressingboth the sequence of interest and the repressor protein. In one example,cell lines may be made that contain Lenti6/TR and Lenti4/TO/V5-GW/lacZand such cells may show at least 20-fold induction.

[1210] One aspect of the present invention is the capability to regulatethe expression of a sequence of interest in a non-dividing cell. In aspecific embodiment, the present invention provides non-dividing hostcells containing a sequence of interest, the expression of which isregulatable, for example, is inducible by the addition of tetracyclineto the growth medium of the cell. The present invention contemplatescompositions comprising such cells and further comprising one or morecomponent selected from a group consisting of an inducer (e.g.,tetracycline), a growth medium, and a buffer.

[1211] A nucleic acid molecule expressing the tetracycline repressorprotein may be constructed using any technique known in the art. Forexample, a nucleic acid fragment containing the tetracycline repressorcoding sequence can be cloned using any technique known in the art. Thenucleotide sequence of a nucleic acid fragment containing the codingsequence for the tetracycline repressor is provided as Table 35. The 1.4kb fragment also contains the β-globin intron. The 1.4 kbTetR-containing fragment was cloned into pLenti6/V5 (InvitrogenCorporation, Carlsbad, Calif.). A map of pLenti6/V5 is provided as FIG.71 and the nucleotide sequence is provided as Table 33. The resultingplasmid, pLenti6/TR, was verified by restriction digest and sequenceanalyses. A map of pLenti6/TR is shown in FIG. 69. pLenti6/TR can beused to generate blasticidin resistant mammalian cells that stablyexpress the tetracycline repressor, TetR.

[1212] Nucleic acid molecules comprising a promoter sequence and arepressor sequence can be constructed using any techniques known in theart. For example, pLenti4/TO/V5-DEST was created from pLenti3/V5-TREx(Invitrogen Corporation, Carlsbad, Calif.), by replacing the neomycinresistance gene of the latter with the zeocin resistance gene.pLenti3/V5-TREx contains the CMV promoter and Tet operators ofpT-REx-DEST30 (Invitrogen Corporation, Carlsbad, Calif. catalog no.12301016). A map of pLenti3/V5-TREx is provided as FIG. 72 and thenucleotide sequence is provided in Table 34.

[1213] pLenti3/V5/TREx was digested with SalI, filled in using Klenowand then digest with KpnI and the 5917 bp vector backbone was gelisolated. Next, pLenti4/V5-DEST (Invitrogen Corporation, Carlsbad,Calif. catalog nos. K498000 and V49810) was digested with Spel, Klenowfilled-in, then digested with KpnI. A 2682 bp fragment ofpLenti4/V5-DEST containing a GATEWAY™ Destination cassette, SV40promoter and Zeocin resistance cassette, was gel isolated and ligated tothe SalI-Klenow-KpnI processed pLenti3/V5-TREx. The ligation mixture wastransformed into DB3.1 and selected on LB media containing Amp (100μg/ml) and chloramphenicol (15 μg/ml). Colonies of the transformantswere analyzed by restriction analysis. A map of pLenti4/TO/V5-DEST isshown in FIG. 70A. The GATEWAY™ Destination vector pLenti4/TO/V5-DESTcontains the tet-regulated CMVTetO₂ T-REx promoter (consisting of CMVpromoter and two tet operator sites). TetR protein binds the tetO sitesto inhibit gene transcription; tetracycline relieves the transcriptioninhibition. pLenti4/TO/V5-DEST confers zeocin resistance and allowsin-frame fusion of genes-of-interest to the V5 epitope tag.

[1214] pLentiTO/V5-GW/lacZ was generated by standard Gateway LxRreaction between pLenti4/TO/V5-DEST and pENTR/dT-lacZ no stop(Invitrogen Corporation, Carlsbad, Calif.). Clones ofpLenti4/TO/V5-GW/lacZ were confirmed by restriction and sequenceanalyses. A map of pLenti4/TO/V5-GW/lacZ is shown in FIG. 70B.

[1215] 293FT (Invitrogen Corporation, Carlsbad, Calif. catalog no.R70007) and GripTite 293 (Invitrogen Corporation, Carlsbad, Calif.catalog no. R79507) cells a were cultured in DMEM/10%FBS/L-glutamine/non-essential amino acids/penicillin/streptomycincontaining 500 μg/ml G418. MJ90 primary human foreskin fibroblasts(Grand Island) and HT1080 human fibrosarcoma (ATCC #CCL-121) werecultured in DMEM/10% FBS/non-essential aminoacids/penicillin/streptomycin. 10 μg/ml blasticidin was used to selectfor stable pLenti6/TR-transduced cells. MJ90 primary cells were growtharrested by contact inhibition. Briefly, 1×10⁵ cells were plated perwell of a 6-well plate and media changes were performed every 3 days for7 to 14 days, or until a quiescent monolayer was achieved.

[1216] For virus production, 5×10⁶ 293FT cells were plated per 100 mmplate. Twenty-four hours later, the culture medium was replaced with 5ml OptiMem/10% FBS and cells were quadruple co-transfected, as follows.12 μg DNA total, at a mass ratio of 1:1:1:1 pLenti6/TR orpLentiTO/V5-GW/lacZ :pLP1:LP2:pLP/VSVG (3 μg of each DNA) was mixed with1.5 ml of OptiMem media. In a separate tube, 36 μl of Lipofectamine 2000was also mixed with 1.5 ml of OptiMem media. After a 5-minute incubationperiod at room temperature, the two mixtures were combined and incubatedat room temperature for an additional 20 minutes. At the completion ofthe incubation period, the transfection mixture was added to the cellsdropwise and the culture plate was gently swirled to mix. The followingday the transfection complex was replaced with complete media (DMEM, 10%FBS, 1% penicillin/streptomycin, L-glutamine and non-essential aminoacids). Forty-eight to seventy-two hours post transfection, thevirus-containing supernatants were harvested, centrifuged at 3000 rpmfor 5 minutes to remove dead cells and placed in cryovials in 1 mlaliquots. Titers were performed on fresh supernatants (see below) andthe remaining viral aliquots were stored at −70° C.

[1217] All applications of virus to cells were performed in the presenceof 6 μg/ml polybrene (Sigma #H9268) and media changes were performed12-24 hours post transduction. For titering virus, 6-well plates wereseeded at 2×10⁵ cells per well with HT1080 cells the day beforetransduction. One well served as an untransduced control (mock) and theremaining five wells contained 1 ml each of ten-fold serial dilutions ofviral supernatant ranging from 10⁻² to 10³¹ ⁶. The dilutions were mixedby gentle inversion prior to adding to cells. 6 μg/ml of polybrene wasadded to each well. The plate was gently swirled to mix. The followingday, the media was replaced with complete media. Forty-eight hourspost-transduction, the cells were placed under 10 μg/ml blasticidin or100 μg/ml zeocin selection, as appropriate. In particular, Zeocinselection was done as follows: 24-hour post-transduction cells weretrypsinized from 6-well plates and expanded into 100 mm plates. 24 hrsafter expansion into 100 mm plates, 100 μg/ml Zeocin was added to thetransduced cell culture medium for selection. After 7 to 10 days ofblasticidin selection, or two-to-three weeks of zeocin selection, theresulting colonies were stained with crystal violet: A 1% crystal violetsolution was prepared in 10% ethanol. Each well was washed with 2 ml PBSfollowed by 1 ml of crystal violet solution for 10 minutes at roomtemperature. Excess stain was removed by two 2 ml PBS washes andcolonies visible to the naked eye were counted to determine the viraltiter of the original supernatants.

[1218] Cell lysates for western blot and Tropix Assays were prepared asfollows: Culture media were aspirated and cells were washed 1× with PBSand followed by incubation in Versene (Invitrogen Corporation, Carlsbad,Calif. catalog no. 15040066) for 2 minutes at room temperature. Detachedcells were pelleted in Eppendorf Tubes and lysed in ice-cold 100 μlNP-40 lysis buffer (50 mM Tris, 150 mM NaCl, 1% NP-40, pH 8.0)containing protease inhibitors. Lysates were centrifuiged at 14000 rpmfor 5 min to pellet cellular debris; the supernatant was collected andfrozen at −70° C. until needed for assays. Protein concentrations weredetermined using BioRad Protein Assay protocol according to themanufacturer's (Biorad) recommendations.

[1219] Western blots were performed using 20 μg of normalized protein ina 4× loading dye. Samples were run on a Novex^(R) Tris-Glycine 4-20% Gel(Invitrogen Corporation, Carlsbad, Calif. catalog no. EC60252BOX), at200 volts for 45 minutes. Protein was transferred to a nitrocellulosemembrane and were detected with a WesternBreeze^(R) Chemiluminescent Kit(Invitrogen Corporation, Carlsbad, Calif. catalog no. WB7104) usingpolyclonal anti-TetR and monoclonal anti-V5 (Invitrogen Corporation,Carlsbad, Calif. catalog no. R96025) primary antibodies, as appropriate.

[1220] pLenti6/TR and pLenti4/TO/V5-GW/lacZ were transfected into 293FTcells, in the presence of Virapower Packaging Mix, to produce therespective viruses. pLenti6/TR and pLenti4/TO/V5-GW/lacZ produced viraltiters of 6×10⁵ and 1×10⁵ cfu/ml respectively. Thus introduction ofβ-globin intron and TetR into pLenti6, and introduction of Tet Operatorsinto pLenti4-DEST, do not compromise virus packaging and transductionefficiency.

[1221] Materials and methods of the invention may be used for a widevariety of purposes. For example, a nucleic acid molecule expressing arepressor protein (e.g., Lenti6/TR virus) may be used to generaterepressor expressing cell lines. Such cell lines may be transduced witha nucleic acid molecule comprising promoter and repressor sequencesoperably linked to a sequence of interest (e.g., Lenti4/TO/5-GW/sequenceof interest) and then expression of the sequence of interest may beregulated (e.g., using tetracycline). Another use of the materials andmethods of the invention is to simultaneously cotransduce a nucleic acidmolecule encoding a repressor and a nucleic acid molecule comprisingpromoter and repressor sequences operably linked to sequence of interest(e.g., Lenti6/TR and Lenti4/TO/V5-GW/sequence of interest) into primarynon-dividing cells, then regulate expression of the sequence of interest(e.g., using tetracycline).

[1222] HT1080 cells were transduced with Lenti6/TR virus at MOI of 1, 10or 32 and were selected in blasticidin medium until mock transducedcells had died-off. The blasticidin-resistant cells were next transducedwith Lenti4/TO/V5-GW/lacZ virus at MOI of 5. Twenty-four hours aftertransducing with Lenti4/TO/V5-GW/lacZ, 1 μg/ml tetracycline was added tothe culture medium. Cells were incubated in the inducer-supplementedmedium for 48 hrs. Thereafter, cell lysates were prepared and analyzedfor gene expression by (i) assaying for lacZ activity; (ii) performingwestern blot for lacZ-V5 fusion using anti-V5 antibody; (iii) westernblot for TetR.

[1223] Increasing the amount of transduced TetR virus reduced lacZexpression in the absence of tetracycline. Tetracycline at 1 μg/mlinduces lacZ expression to levels approaching full-strength CMV promoteractivity. To determine fold induction, the ratios of β-galactosidaseactivities in the presence and absence of tetracycline (for a given MOI)were calculated. Induction of lacZ expression was 4-, 17- and 27-fold atTetR MOI of 1, 10 and 32, respectively, indicating that induction wasdependent on the amount of TetR. Western blot analyses using anti-V5antibody was consistent with the β-galactosidase enzymatic activitydata. Expression of TetR protein was confirmed by western blot usingpolyclonal anti-TetR antibody.

[1224] These results confirm CMVTetO₂ and TetR in the lentiviral vectorsto be functional and responsive to tetracycline. The relatively highlevel basal transcription from CMVTetO₂ at lower Lenti6/TR MOIs couldresult from the fact that not all blasticidin resistant cells generatedat the low TetR MOIs actually express TetR. Those cells that do notexpress TetR would express lacZ from CMVTetO₂ promoter withoutinhibition and produce a high background. By contrast, at high Lenti/TRMOIs, close to 100% of blasticidin-resistant cells generated wouldexpress TetR, inhibit transcription from CMVTetO₂ promoter and producelower background lacZ expression.

[1225] The data in HT1080 cells showed that lower basal transcription ina cell population is achieved at higher TetR levels. Therefore whentesting induction in GripTite 293 cells, Lenti6/TR was transduced atMOI=10 and MOI=32 to generate blasticidin-resistant GripTite-10 andGripTite-32 populations, respectively. These populations were transducedwith Lenti4/TO/V5-GW/lacZ virus at MOI=1 or MOI=5 and tested for lacZinduction. Tetracycline was used at 1 μg/ml or at 5 μg/ml to determineif inducer was limiting at higher TetR concentrations.

[1226] TetR effectively inhibited lacZ expression in GripTite-10 cellsin the absence of inducer and this repression was relieved bytetracycline. 1 μg/ml tetracycline was nearly as effective as 5 μg/mltetracycline in inducing gene expression. Fold induction was calculatedas induced:uninduced ratios at a given Lenti4/TO/V5-GW/lacZ MOI andtetracycline concentration. LacZ expression was induced over 27-fold atLenti4/TO/V5-GW/lacZ MOI=5 compared to just above 7-fold atLenti4/TO/V5-GW/lacZ MOI=1. Western blot analyses using anti-VS antibodyreflected β-gal enzymatic Tropix data. Expression of TetR protein wasconfirmed by western blot using polyclonal anti-TetR antibody.

[1227] The results in GripTite 293-10 cells were recapitulated inGripTite-32 cells. As in GripTite 293-10 cells, 1 μg/ml was nearly aseffective as 5 μg/ml tetracycline in inducing gene expression inGripTite-32 cells. The fold lacZ induction was significantly higher inGripTite 293-32 cells however and ranged from 57 to 72 fold atLenti4/TO/V5-GW/lacZ MOI=1 and Lenti4/TO/V5-GW/lacZ MOI=5, respectively.

[1228] The data indicate that 1 μg/ml tetracycline is not limiting ininducing lacZ expression. LacZ induction was higher atLenti4/TO/V5-GW/lacZ MOI=5 than at MOI=1. Thus the amount of expressionmay be adjusted by altering the MOI of the virus containing a sequenceof interest operably linked to a promoter and repressor sequence (e.g.,higher MOI for higher expression level when de-repressed, lower MOI forlower expression level when de-repressed). The increased MOI has littleeffect on background uninduced levels when TetR is not limiting (e.g.,MOI of 10 and 32).

[1229] In one particular embodiment, materials and methods of theinvention may be used to regulate gene expression in non-dividingprimary cells. MJ90 cells are contact-inhibited primary fibroblasts thatundergo growth arrest at confluence and are refractory to both lipidtransfection and transduction by Moloney retroviral vectors. MJ90 cellswere transduced with 2×10⁶ cfu/well Lenti6/TR virus for 24 hrs followedby transduction with 2×10⁶ cfu/well of Lenti4/TO/V5-GW/lacZ virus(estimated MOI=7.5 each). Twenty-four hours after transducing withLenti4/TO/V5-GW/lacZ, lacZ expression was induced with 1 μg/mltetracycline for 48 hrs. Lysates from transduced cells were analyzed forprotein induction. TetR repressed expression of lacZ over 90%, resultingin a 10-fold induction. It is worth noting that the preceding experimentwas performed with equal MOI of Lenti6/TR and Lenti4/TO/V5-GW/lacZ.Higher Lenti6/TR MOI, or different Lenti6/TR: Lenti4/TO/V5-GW/lacZratios may be used to give higher inducibility. The demonstration thatthe present invention can regulate gene expression in quiescent primarycells is significant especially since the cells are hard to transfectand resist transduction by Moloney retroviral vectors.

[1230] Nucleic acid molecules of the invention comprising promoter andrepressor sequences operably linked to a sequence of interest may beused in conjunction with any nucleic acid molecule expressing arepressor protein. For example, Lenti4/TO/V5-GW/lacZ virus wastransduced into the Flp-In T-REx 293 product cell line (InvitrogenCorporation, Carlsbad, Calif. catalog no. R78007) at MOIs of 1 and 2.5.Gene expression was induced with 1 μg/ml tetracycline for 48 hrs.Tetracycline induced lacZ expression from Lenti4/TO/V5-GW/lacZ in Flp-InT-REx 293 cells. Increasing the amount of transducedLenti4/TO/V5-GW/lacZ from MOI=1 to MOI=2.5 increased induction from16-fold to 24-fold, respectively similar to the results in GripTite-10and GripTite-32 populations.

Example 18

[1231] In some embodiments, the present invention provides a method ofcovalently attaching an enzyme (e.g., a topoisomerase) to a nucleic acidmolecule. In one aspect, a nucleic acid molecule for use in methods ofthis type may comprise a restriction enzyme recognition sequence (e.g.,a TypeIls restriction enzyme recognition) and a topoisomeraserecognition sequence. In some embodiments, a TypeIIs recognitionsequence may be located adjacent to a topoisomerase recognitionsequence. In this regard, adjacent means that the cleavage sites of thetwo enzymes may be within from about 1 to about 50, from about 1 toabout 45, from about 1 to about 40, from about 1 to about 35, from about1 to about 30, from about 1 to about 25, from about 1 to about 20, fromabout 1 to about 15, from about 1 to about 10, from about 1 to about 9,from about 1 to about 8, from about 1 to about 7, from about 1 to about6, from about 1 to about 5, from about 1 to about 4, from about 1, toabout 3, or from about 1 to about 2 base pairs from each other. AnyTypeIIs enzyme may be used. In some embodiments, a suitable TypeIIsenzyme may leave a 3′-overhanging sequence. Suitable TypeIIs enzymesinclude Bael.

[1232] With reference to FIG. 73, a nucleic acid molecule of theinvention may comprise two topoisomerase recognition sites and twoTypeIIs recognition sites, for example with the two restriction enzymesites between the two topisomerase sites. Optionally a nucleic acidmolecule of this type may comprise nucleic acid sequence between therestriction enzyme sites. The nucleic acid sequence between therestriction enzyme sites may encode a polypeptide, for example, aselectable marker such as the ccdB gene.

[1233] The restriction enzyme sites may be located such that a3′-overhang of a desired length is produced on the strand containing thetopoisomerase cleavage site (after the 3′-T in FIG. 73). The location ofthe topoisomerase cleavage site may be varied with respect to 3′-mostnucleotide of the strand containing the cleavage site. This may beuseful in generating a 5′-overhang on the opposite strand aftertopoisomerase cleavage in order to generate a sequence that can invade adouble-stranded insert (see FIG. 47).

[1234] After restriction enzyme cleavage, the cleaved vector may becontacted with an oligonucleotide that anneals to the 3′-overhangingsequence and/or may be contacted with a topoisomerase.

[1235] In some embodiments, methods of the invention may comprisedigesting a nucleic acid molecule of the invention (e.g., 20 μg) with aTypeIIs restriction enzyme (e.g., 100 Units of BaeI, New EnglandBiolabs, catalog no. R0613S), for example, in a final volume of 250 μl.Any other restriction enzyme known in the art may be also be used. Thereaction may be carried out in a suitable buffer (e.g., 1×NEBuffer 2with 100 μg/ml of BSA and 20 μM S-adenosylmethionine, New EnglandBiolabs) under suitable conditions (e.g., at 37° C. for 6 hours). Thedigestion may be terminated, for example, with the addition of 250 μl ofPhenol/Chloroform (Invitrogen Corporation, Carlsbad, Calif., Cat.#15593-031) and mixing. The organic and aqueous phases may be separatedby centrifugation at 14,000×g at 4° C. for 5 minutes. The aqueous (top)layer may be transferred to a new tube and 25 μl of 3M sodium acetate(pH 5.2) may be added and mixed. This may be followed by 625 μl of 100%ethanol and incubation in ice for 5 minutes. Precipitated DNA may be washarvested by centrifugation at 14,000×g for 5 minutes at 4° C. The DNApellet may be washed with 500 μl of 70% ethanol, harvested bycentrifugation at 14,000×g for 5 minutes at 22° C. The pellet may beallowed to dry and then resuspended in 100 μl of TE. The DNAconcentration may be determined by its optical density at 260 nm.

[1236] The digested vector may be contacted with an oligonucleotide thatanneals to all or a portion of the 3′-overhang produced by therestriction enzyme and/or with a suitable topoisomerase enzyme (e.g.,Vaccinia DNA Topoisomerase) in a suitable buffer (e.g., 1×NEBuffer #1,New England Biolabs), for example, in a final volume of 50 μl. Thereaction may be incubated under suitable conditions (e.g., 25° C. for 15minutes). Then reaction may be terminated with the addition of 5 μl of10×Stop Buffer. The topoisomerase-linked vector may be purified by gelelectrophoresis (see, Heyman, et al. Genome Research 9:383-392 (1999)).

[1237] Having now fully described the present invention in some detailby way of illustration and example for purposes of clarity ofunderstanding, it will be obvious to one of ordinary skill in the artthat the same can be performed by modifying or changing the inventionwithin a wide and equivalent range of conditions, formulations and otherparameters without affecting the scope of the invention or any specificembodiment thereof, and that such modifications or changes are intendedto be encompassed within the scope of the appended claims.

[1238] All publications, patents and patent applications mentioned inthis specification are indicative of the level of skill of those skilledin the art to which this invention pertains, and are herein incorporatedby reference to the same extent as if each individual publication,patent or patent application was specifically and individually indicatedto be incorporated by reference. TABLE 6 Nucleotide sequence ofpAd/CMV/V5-DEST.catcatcaataatataccttattttggattgaagccaatatgataatgagggggtggagtttgtgacgtggcgcggggcgtgggaacggggcgggtgacgtagtagtgtggcggaagtgtgatgttgcaagtgtggcggaacacatgtaagcgacggatgtggcaaaagtgacgtttttggtgtgcgccggtgtacacaggaagtgacaattttcgcgcggttttaggcggatgttgtagtaaatttgggcgtaaccgagtaagatttggccattttcgcgggaaaactgaataagaggaagtgaaatctgaataattttgtgttactcatagcgcgtaatatttgtctagggccgcggggactttgaccgtttacgtggagactcgcccaggtgtttttctcaggtgttttccgcgttccgggtcaaagttggcgttttattattatagtcagtcgaagcttggatccggtacctctagaattctcgagcggccgctagcgacatcggatctcccgatcccctatggtcgactctcagtacaatctgctctgatgccgcatagttaagccagtatctgctccctgcttgtgtgttggaggtcgctgagtagtgcgcgagcaaaatttaagctacaacaaggcaaggcttgaccgacaattgcatgaagaatctgcttagggttaggcgttttgcgctgcttcgcgatgtacgggccagatatacgcgttgacattgattattgactagttattaatagtaatcaattacggggtcattagttcatagcccatatatggagttccgcgttacataacttacggtaaatggcccgcctggctgaccgcccaacgacccccgcccattgacgtcaataatgacgtatgttcccatagtaacgccaatagggactttccattgacgtcaatgggtggactatttacggtaaactgcccacttggcagtacatcaagtgtatcatatgccaagtacgccccctattgacgtcaatgacggtaaatggcccgcctggcattatgcccagtacatgaccttatgggactttcctacttggcagtacatctacgtattagtcatcgctattaccatggtgatgcggttttggcagtacatcaatgggcgtggatagcggtttgactcacggggatttccaagtctccaccccattgacgtcaatgggagtttgttttggcaccaaaatcaacgggactttccaaaatgtcgtaacaactccgccccattgacgcaaatgggcggtaggcgtgtacggtgggaggtctatataagcagagctctctggctaactagagaacccactgcttactggcttatcgaaattaatacgactcactatagggagacccaagctggctagttaagctatcaacaagtttgtacaaaaaagctgaacgagaaacgtaaaatgatataaatatcaatatattaaattagattttgcataaaaaacagactacataatactgtaaaacacaacatatccagtcactatgaatcaactacttagatggtattagtgacctgtagtcgaccgacagccttccaaatgttcttcgggtgatgctgccaacttagtcgaccgacagccttccaaatgttcttctcaaacggaatcgtcgtatccagcctactcgctattgtcctcaatgccgtattaaatcataaaaagaaataagaaaaagaggtgcgagcctcttttttgtgtgacaaaataaaaacatctacctattcatatacgctagtgtcatagtcctgaaaatcatctgcatcaagaacaatttcacaactcttatacttttctcttacaagtcgttcggcttcatctggattttcagcctctatacttactaaacgtgataaagtttctgtaatttctactgtatcgacctgcagactggctgtgtataagggagcctgacatttatattccccagaacatcaggttaatggcgtttttgatgtcattttcgcggtggctgagatcagccacttcttccccgataacggagaccggcacactggccatatcggtggtcatcatgcgccagctttcatccccgatatgcaccaccgggtaaagttcacgggagactttatctgacagcagacgtgcactggccagggggatcaccatccgtcgcccgggcgtgtcaataatatcactctgtacatccacaaacagacgataacggctctctcttttataggtgtaaaccttaaactgcatttcaccagtccctgttctcgtcagcaaaagagccgttcatttcaataaaccgggcgacctcagccatcccttcctgattttccgctttccagcgttcggcacgcagacgacgggcttcattctgcatggttgtgcttaccagaccggagatattgacatcatatatgccttgagcaactgatagctgtcgctgtcaactgtcactgtaatacgctgcttcatagcacacctctttttgacatacttcgggtatacatatcagtatatattcttataccgcaaaaatcagcgcgcaaatacgcatactgttatctggcttttagtaagccggatccacgcgattacgccccgccctgccactcatcgcagtactgttgtaattcattaagcattctgccgacatggaagccatcacagacggcatgatgaacctgaatcgccagcggcatcagcaccttgtcgccttgcgtataatatttgcccatggtgaaaacgggggcgaagaagttgtccatattggccacgtttaaatcaaaactggtgaaactcacccagggattggctgagacgaaaaacatattctcaataaaccctttagggaaataggccaggttttcaccgtaacacgccacatcttgcgaatatatgtgtagaaactgccggaaatcgtcgtggtattcactccagagcgatgaaaacgtttcagtttgctcatggaaaacggtgtaacaagggtgaacactatcccatatcaccagctcaccgtctttcattgccatacggaattccggatgagcattcatcaggcgggcaagaatgtgaataaaggccggataaaacttgtgcttatttttctttacggtctttaaaaaggccgtaatatccagctgaacggtctggttataggtacattgagcaactgactgaaatgcctcaaaatgttctttacgatgccattgggatatatcaacggtggtatatccagtgatttttttctccattttagcttccttagctcctgaaaatctcgataactcaaaaaatacgcccggtagtgatcttatttcattatggtgaaagttggaacctcttacgtgccgatcaacgtctcattttcgccaaaagttggcccagggcttcccggtatcaacagggacaccaggatttatttattctgcgaagtgatcttccgtcacaggtatttattcggcgcaaagtgcgtcgggtgatgctgccaacttagtcgactacaggtcactaataccatctaagtagttgattcatagttttcttgtacaaagtggttgatctagagggcccgcggttcgaaggtaagcctatccctaaccctctcctcggtctcgattctacgcgtaccggttagtaatgagtttaaacgggggaggctaactgaaacacggaaggagacaataccggaaggaacccgcgctatgacggcaataaaaagacagaataaaacgcacgggtgttgggtcgtttgttcataaacgcggggttcggtcccagggctggcactctgtcgataccccaccgagaccccattggggccaatacgcccgcgtttcttccttttccccaccccaccccccaagttcgggtgaaggcccagggctcgcagccaacgtcggggcggcaggccctgccatagcagatccgattcgacagatcactgaaatgtgtgggcgtggcttaagggtgggaaagaatatataaggtgggggtcttatgtagttttgtatctgttttgcagcagccgccgccgccatgagcaccaactcgtttgatggaagcattgtgagctcatatttgacaacgcgcatgcccccatgggccggggtgcgtcagaatgtgatgggctccagcattgatggtcgccccgtcctgcccgcaaactctactaccttgacctacgagaccgtgtctggaacgccgttggagactgcagcctccgccgccgcttcagccgctgcagccaccgcccgcgggattgtgactgactttgctttcctgagcccgcttgcaagcagtgcagcttcccgttcatccgcccgcgatgacaagttgacggctcttttggcacaattggattctttgacccgggaacttaatgtcgtttctcagcagctgttggatctgcgccagcaggtttctgccctgaaggcttcctcccctcccaatgcggtttaaaacataaataaaaaaccagactctgtttggatttggatcaagcaagtgtcttgctgtctttatttaggggttttgcgcgcgcggtaggcccgggaccagcggtctcggtcgttgagggtcctgtgtattttttccaggacgtggtaaaggtgactctggatgttcagatacatgggcataagcccgtctctggggtggaggtagcaccactgcagagcttcatgctgcggggtggtgttgtagatgatccagtcgtagcaggagcgctgggcgtggtgcctaaaaatgtctttcagtagcaagctgattgccaggggcaggcccttggtgtaagtgtttacaaagcggttaagctgggatgggtgcatacgtggggatatgagatgcatcttggactgtatttttaggttggctatgttcccagccatatccctccggggattcatgttgtgcagaaccaccagcacagtgtatccggtgcacttgggaaatttgtcatgtagcttaga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cacgaaccccccgttcagcccgaccgctgcgccttatccggtaactatcgtcttgagtccaacccggtaagacacgacttatcgccactggcagcagccactggtaacaggattagcagagcgaggtatgtaggcggtgctacagagttcttgaagtggtggcctaactacggctacactagaaggacagtatttggtatctgcgctctgctgaagccagttaccttcggaaaaagagttggtagctcttgatccggcaaacaaaccaccgctggtagcggtggtttttttgtttgcaagcagcagattacgcgcagaaaaaaaggatctcaagaagatcctttgatcttttctacggggtctgacgctcagtggaacgaaaactcacgttaagggattttggtcatgagattatcaaaaaggatcttcacctagatccttttaaatcaatctaaagtatatatgagtaaacttggtctgacagttaccaatgcttaatcagtgaggcacctatctcagcgatctgtctatttcgttcatccatagttgcctgactccccgtcgtgtagataactacgatacgggagggcttaccatctggccccagtgctgcaatgataccgcgagacccacgctcaccggctccagatttatcagcaataaaccagccagccggaagggccgagcgcagaagtggtcctgcaactttatccgcctccatccagtctattaattgttgccgggaagctagagtaagtagttcgccagttaatagtttgcgcaacgttgttgccattgntgcaggcatcgtggtgtcacgctcgtcgtttggtatggcttcattcagctccggttcccaacgatcaaggcgagttacatgatcccccatgttgtgcaaaaaagcggttagctccttcggtcctccgatcgttgtcagaagtaagttggccgcagtgttatcactcatggttatggcagcactgcataattctcttactgtcatgccatccgtaagatgcttttctgtgactggtgagtactcaaccaagtcattctgagaatagtgtatgcggcgaccgagttgctcttgcccggcgtcaacacgggataataccgcgccacatagcagaactttaaaagtgctcatcattggaaaacgttcttcggggcgaaaactctcaaggatcttaccgctgttgagatccagttcgatgtaacccactcgtgcacccaactgatcttcagcatcttttactttcaccagcgtttctgggtgagcaaaaacaggaaggcaaaatgccgcaaaaaagggaataagggcgacacggaaatgttgaatactcatactcttcctttttcaatattattgaagcatttatcagggttattgtctcatgagcggatacatatttgaatgtatttagaaaaataaacaaataggggttccgcgcacatttccccgaaaagtgccacctgacgtctaagaaaccattattatcatgacattaacctataaaaataggcgtatcacgaggccctttcgtcttcaaggatccgaattcccgggagagctcgatatcgcatgcggatttaaattaattaa

[1239] TABLE 7 Nucleotide sequence of pAd-GW-TO/tRNA.catcatcaataatataccttattttggattgaagccaatatgataatgagggggtggagtttgtgacgtggcgcggggcgtgggaacggggcgggtgacgtagtagtgtggcggaagtgtgatgttgcaagtgtggcggaacacatgtaagcgacggatgtggcaaaagtgacgtttttggtgtgcgccggtgtacacaggaagtgacaattttcgcgcggttttaggcggatgttgtagtaaatttgggcgtaaccgagtaagatttggccattttcgcgggaaaactgaataagaggaagtgaaatctgaataattttgtgttactcatagcgcgtaatatttgtctagggccgcggggactttgaccgtttacgtggagactcgcccaggtgtttttctcaggtgttttccgcgttccgggtcaaagttggcgttttattattatagtcagtcgaagcttggatccggtacctctagaattctcgagcggccgctagcgacatcgatcacaagtttgtacaaaaaagcaggctttaaaggaaccaattcagtcgactctagaggatcgaaaccatcctctgctatatggccgcatatattttacttgaagactaggaccctacagaaaaggggttttaaagtaggcgtgctaaacgtcagcggacctgacccgtgtaagaatccacaaggtatcctggtggaaatgcgcatttgtaggcttcaatatctgtaatcctactaattaggtgtggagagctttcagccagtttcgtaggtttggagaccatttaggggttggcgtgtggccccctcgtaaagtctttcgtacttcctacatcagacaagtcttgcaatttgcaatatctcttttagccaatatctaaatctttaaaattttgattttgttttttacccaggatgagagacattccagagttgttaccttgtcaaaataaacaaatttaaagatgtctgtgaaaagaaacatatattcctcatgggaatatatccaggttgttgaaggaggtacgacctcgagatctctatcactgatagggagactcgagtgtagtcgtggccgagtggttaaggcgatggactctaaatccattggggtctccccgcgcaggttcgaatcctgccgactacggcgtgctttttttactctcgggtagaggaaatccggtgcactacctgtgcaatcacacagaataacatggagtagtactttttattttcctgttattatctttctccataaaagtggaaccagataattttagttcttttgtgtaacaagactagagattttttgaagtgttacattggaaagcacttgaaaacacaagtaatttctgacactgctataaaaatgatggaaaaacgctcaagttgttttgcctttcagtcttcttgaaatgctgtctccctatctgaaatccagctcacgtctgacttccaaaaccgtgcttgcctttaacttatggaataaatatctcaaacagatccccgggcgagctcgaattcgcggccgcactcgagatatctagacccagctttcttgtacaaagtggtgatcgattcgacagatcactgaaatgtgtgggcgtggcttaagggtgggaaagaatatataaggtgggggtcttatgtagttttgtatctgttttgcagcagccgccgccgccatgagcaccaactcgtttgatggaagcattgtgagctcatatttgacaacgcgcatgcccccatgggccggggtgcgtcagaatgtgatgggctccagcattgatggtcgccccgtcctgcccgcaaactctactaccttgacctacgagaccgtgtctggaacgccgttggagactgcagcctccgccgccgcttcagccgctgcagccaccgcccgcgggattgtgactgactttgctttcctgagcccgcttgcaagcagtgcagcttcccgttcatccgcccgcgatgacaagttgacggctcttttggcacaattggattctttgacccgggaacttaatgtcgtttctcagcagctgttggatctgcgccagcaggtttctgccctgaaggcttcctcccctcccaatgcggtttaaaacataaataaaaaaccagactctgtttggatttggatcaagcaagtgtcttgctgtctttatttaggggttttgcgcgcgcggtaggcccgggaccagcggtctcggtcgttgagggtcctgtgtattttttccaggacgtggtaaaggtgactctggatgttcagatacatgggcataagcccgtctctggggtggaggtagcaccactgcagagcttcatgctgcggggtggtgttgtagatgatccagtcgtagcaggagcgctgggcgtggtgcctaaaaatgtctttcagtagcaagctgattgccaggggcaggcccttggtgtaagtgtttacaaagcggttaagctgggatgggtgcatacgtggggatatgagatgcatcttggactgtatttttaggttggctatgttcccagccatatccctccggggattcatgttgtgcagaaccaccagcacagtgtatccggtgcacttgggaaatttgtcatgtagcttagaaggaaatgcgtggaagaacttggagacgcccttgtgacctccaagattttccatgcattcgtccataatgatggcaatgggcccacgggcggcggcctgggcgaagatatttctgggatcactaacgtcatagttgtgttccaggatgagatcgtcataggccatttttacaaagcgcgggcggagggtgccagactgcggtataatggttccatccggcccaggggcgtagttaccctcacagatttgcatttcccacgctttgagttcagatggggggatcatgtctacctgcggggcgatgaagaaaacggtttccggggtaggggagatcagctgggaagaaagcaggttcctgagcagctgcgacttaccgcagccggtgggcccgtaaatcacacctattaccgggtgcaactggtagttaagagagctgcagctgccgtcatccctgagcaggggggccacttcgttaagcatgtccctgactcgcatgttttccctgaccaaatccgccagaaggcgctcgccgcccagcgatagcagttcttgcaaggaagcaaagtttttcaacggtttgagaccgtccgccgtaggcatgcttttgagcgtttgaccaagcagttccaggcggtcccacagctcggtcacctgctctacggcatctcgatccagcatatctcctcgtttcgcgggttggggcggctttcgctgtacggcagtagtcggtgctcgtccagacgggccagggtcatgtctttccacgggcgcagggtcctcgtcagcgtagtctgggtcacggtgaaggggtgcgctccgggctgcgcgctggccagggtgcgcttgaggctggtcctgctggtgctgaagcgctgccggtcttcgccctgcgcgtcggccaggtagcatttgaccatggtgtcatagtccagcccctccgcggcgtggcccttggcgcgcagcttgcccttggaggaggcgccgcacgaggggcagtgcagacttttgagggcgtagagcttgggcgcgagaaataccgattccggggagtaggcatccgcgccgcaggccccgcagacggtctcgcattccacgagccaggtgagctctggccgttcggggtcaaaaaccaggtttcccccatgctttttgatgcgtttcttacctctggtttccatgagccggtgtccacgctcggtgacgaaaaggctgtccgtgtccccgtatacagacttgagaggcctgtcctcgagcggtgttccgcggtcctcctcgtatagaaactcggaccactctgagacaaaggctcgcgtccaggccagcacgaaggaggctaagtgggaggggtagcggtcgttgtccactagggggtccactcgctccagggtgtgaagacacatgtcgccctcttcggcatcaaggaaggtgattggtttgtaggtgtaggccacgtgaccgggtgttcctgaaggggggctataaaagggggtgggggcgcgttcgtcctcactctcttccgcatcgctgtctgcgagggccagctgttggggtgagtactccctctgaaaagcgggcatgacttctgcgctaagattgtcagtttccaaaaacgaggaggatttgatattcacctggcccgcggtgatgcctttgagggtggccgcatccatctggtcagaaaagacaatctttttgttgtcaagcttggtggcaaacgacccgtagagggcgttggacagcaacttggcgatggagcgcagggtttggtttttgtcgcgatcggcgcgctccttggccgcgatgtttagctgcacgtattcgcgcgcaacgcaccgccattcgggaaagacggtggtgcgctcgtcgggcaccaggtgcacgcgccaaccgcggttgtgcagggtgacaaggtcaacgctggtggctacctctccgcgtaggcgctcgttggtccagcagaggcggccgcccttgcgcgagcagaatggcggtagggggtctagctgcgtctcgtccggggggtctgcgtccacggtaaagaccccgggcagcaggcgcgcgtcgaagtagtctatcttgcatccttgcaagtctagcgcctgctgccatgcgcgggcggcaagcgcgcgctcgtatgggttgagtgggggaccccatggcatggggtgggtgagcgcggaggcgtacatgccgcaaatgtcgtaaacgtagaggggctctctgagtattccaagatatgtagggtagcatcttccaccgcggatgctggcgcgcacgtaatcgtatagttcgtgcgagggagcgaggaggtcgggaccgaggttgctacgggcgggctgctctgctcggaagactatctgcc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cacgtgcatacacttcctcaggattacaagctcctcccgcgttagaaccatatcccagggaacaacccattcctgaatcagcgtaaatcccacactgcagggaagacctcgcacgtaactcacgttgtgcattgtcaaagtgttacattcgggcagcagcggatgatcctccagtatggtagcgcgggtttctgtctcaaaaggaggtagacgatccctactgtacggagtgcgccgagacaaccgagatcgtgttggtcgtagtgtcatgccaaatggaacgccggacgtagtcatatttcctgaagcaaaaccaggtgcgggcgtgacaaacagatctgcgtctccggtctcgccgcttagatcgctctgtgtagtagttgtagtatatccactctctcaaagcatccaggcgccccctggcttcgggttctatgtaaactccttcatgcgccgctgccctgataacatccaccaccgcagaataagccacacccagccaacctacacattcgttctgcgagtcacacacgggaggagcgggaagagctggaagaaccatgtttttttttttattccaaaagattatccaaaacctcaaaatgaagatctattaagtgaacgcgctcccctccggtggcgtggtcaaactctacagccaaagaacagataatggcatttgtaagatgttgcacaatggcttccaaaaggcaaacggccctcacgtccaagtggacgtaaaggctaaacccttcagggtgaatctcctctataaacattccagcaccttcaaccatgcccaaataattctcatctcgccaccttctcaatatatctctaagcaaatcccgaatattaagtccggccattgtaaaaatctgctccagagcgccctccaccttcagcctcaagcagcgaatcatgattgcaaaaattcaggttcctcacagacctgtataagattcaaaagcggaacattaacaaaaataccgcgatcccgtaggtcccttcgcagggccagctgaacataatcgtgcaggtctgcacggaccagcgcggccacttccccgccaggaaccttgacaaaagaacccacactgattatgacacgcatactcggagctatgctaaccagcgtagccccgatgtaagctttgttgcatgggcggcgatataaaatgcaaggtgctgctcaaaaaatcaggcaaagcctcgcgcaaaaaagaaagcacatcgtagtcatgctcatgcagataaaggcaggtaagctccggaaccaccacagaaaaagacaccatttttctctcaaacatgtctgcgggtttctgcataaacacaaaataaaataacaaaaaaacatttaaacattagaagcctgtcttacaacaggaaaaacaacccttataagcataagacggactacggccatgccggcgtgaccgtaaaaaaactggtcaccgtgattaaaaagcaccaccgacagctcctcggtcatgtccggagtcataatgtaagactcggtaaacacatcaggttgattcacatcggtcagtgctaaaaagcgaccgaaatagcccgggggaatacatacccgcaggcgtagagacaacattacagcccccataggaggtataacaaaattaataggagagaaaaacacataaacacctgaaaaaccctcctgcctaggcaaaatagcaccctcccgctccagaacaacatacagcgcttccacagcggcagccataacagtcagccttaccagtaaaaaagaaaacctattaaaaaaacaccactcgacacggcaccagctcaatcagtcacagtgtaaaaaagggccaagtgcagagcgagtatatataggactaaaaaatgacgtaacggttaaagtccacaaaaaacacccagaaaaccgcacgcgaacctacgcccagaaacgaaagccaaaaaacccacaacttcctcaaatcgtcacttccgttttcccacgttacgtcacttcccattttaagaaaactacaattcccaacacatacaagttactccgccctaaaacctacgtcacccgccccgttcccacgccccgcgccacgtcacaaactccaccccctcattatcatattggcttcaatccaaaataaggtatattattgatgatgttaattaatttaaatccgcatgcgatatcgagctctcccgggaattcggatctgcgacgcgaggctggatggccttccccattatgattcttctcgcttccggcggcatcgggatgcccgcgttgcaggccatgctgtccaggcaggtagatgacgaccatcagggacagcttcacggccagcaaaaggccaggaaccgtaaaaaggccgcgttgctggcgtttttccataggctccgcccccctgacgagcatcacaaaaatcgacgctcaagtcagaggtggcgaaacccgacaggactataaagataccaggcgtttccccctggaagctccctcgtgcgctctcctgttccgaccctgccgcttaccggatacctgtccgcctttctcccttcgggaagcgtggcgctttctcaatgctcacgctgtaggtatctcagttcggtgtaggtcgttcgctccaagctgggctgtgtgcacgaaccccccgttcagcccgaccgctgcgccttatccggtaactatcgtcttgagtccaacccggtaagacacgacttatcgccactggcagcagccactggtaacaggattagcagagcgaggtatgtaggcggtgctacagagttcttgaagtggtggcctaactacggctacactagaaggacagtatttggtatctgcgctctgctgaagccagttaccttcggaaaaagagttggtagctcttgatccggcaaacaaaccaccgctggtagcggtggtttttttgtttgcaagcagcagattacgcgcagaaaaaaaggatctcaagaagatcctttgatcttttctacggggtctgacgctcagtggaacgaaaactcacgttaagggattttggtcatgagattatcaaaaaggatcttcacctagatccttttaaatcaatctaaagtatatatgagtaaacttggtctgacagttaccaatgcttaatcagtgaggcacctatctcagcgatctgtctatttcgttcatccatagttgcctgactccccgtcgtgtagataactacgatacgggagggcttaccatctggccccagtgctgcaatgataccgcgagacccacgctcaccggctccagatttatcagcaataaaccagccagccggaagggccgagcgcagaagtggtcctgcaactttatccgcctccatccagtctattaattgttgccgggaagctagagtaagtagttcgccagttaatagtttgcgcaacgttgttgccattgntgcaggcatcgtggtgtcacgctcgtcgtttggtatggcttcattcagctccggttcccaacgatcaaggcgagttacatgatcccccatgttgtgcaaaaaagcggttagctccttcggtcctccgatcgttgtcagaagtaagttggccgcagtgttatcactcatggttatggcagcactgcataattctcttactgtcatgccatccgtaagatgcttttctgtgactggtgagtactcaaccaagtcattctgagaatagtgtatgcggcgaccgagttgctcttgcccggcgtcaacacgggataataccgcgccacatagcagaactttaaaagtgctcatcattggaaaacgttcttcggggcgaaaactctcaaggatcttaccgctgttgagatccagttcgatgtaacccactcgtgcacccaactgatcttcagcatcttttactttcaccagcgtttctgggtgagcaaaaacaggaaggcaaaatgccgcaaaaaagggaataagggcgacacggaaatgttgaatactcatactcttcctttttcaatattattgaagcatttatcagggttattgtctcatgagcggatacatatttgaatgtatttagaaaaataaacaaataggggttccgcgcacatttccccgaaaagtgccacctgacgtctaagaaaccattattatcatgacattaacctataaaaataggcgtatcacgaggccctttcgtcttcaaggatccgaattcccgggagagctcgatatcgcatgcggatttaaattaattaa

[1240] TABLE 8 Nucleotide sequence of pAdeno TAG tRNA. 1 catcatcaataatatacctt attttggatt gaagccaata tgataatgag ggggtggagt 61 ttgtgacgtggcgcggggcg tgggaacggg gcgggtgacg tagtagtgtg gcggaagtgt 121 gatgttgcaagtgtggcgga acacatgtaa gcgacggatg tggcaaaagt gacgtttttg 181 gtgtgcgccggtgtacacag gaagtgacaa ttttcgcgcg gttttaggcg gatgttgtag 241 taaatttgggcgtaaccgag taagatttgg ccattttcgc gggaaaactg aataagagga 301 agtgaaatctgaataatttt gtgttactca tagcgcgtaa tatttgtcta gggccgcggg 361 gactttgaccgtttacgtgg agactcgccc aggtgttttt ctcaggtgtt ttccgcgttc 421 cgggtcaaagttggcgtttt attattatag tcagtcgaag cttggatccg gtacctctag 481 aattctcgagcggccgctag cgacatcgat cacaagtttg tacaaaaaag caggctttaa 541 aggaaccaattcagtcgact ctagaggatc gaaaccatcc tctgctatat ggccgcatat 601 attttacttgaagactagga ccctacagaa aaggggtttt aaagtaggcg tgctaaacgt 661 cagcggacctgacccgtgta agaatccaca aggtatcctg gtggaaatgc gcatttgtag 721 gcttcaatatctgtaatcct actaattagg tgtggagagc tttcagccag tttcgtaggt 781 ttggagaccatttaggggtt ggcgtgtggc cccctcgtaa agtctttcgt acttcctaca 841 tcagacaagtcttgcaattt gcaatatctc ttttagccaa tatctaaatc tttaaaattt 901 tgattttgttttttacccag gatgagagac attccagagt tgttaccttg tcaaaataaa 961 caaatttaaagatgtctgtg aaaagaaaca tatattcctc atgggaatat atccaggttg 1021 ttgaaggaggtacgacctcg agatctctat cactgatagg gagactcgag tgtagtcgtg 1081 gccgagtggttaaggcgatg gactctaaat ccattggggt ctccccgcgc aggttcgaat 1141 cctgccgactacggcgtgct ttttttactc tcgggtagag gaaatccggt gcactacctg 1201 tgcaatcacacagaataaca tggagtagta ctttttattt tcctgttatt atctttctcc 1261 ataaaagtggaaccagataa ttttagttct tttgtgtaac aagactagag attttttgaa 1321 gtgttacattggaaagcact tgaaaacaca agtaatttct gacactgcta taaaaatgat 1381 ggaaaaacgctcaagttgtt ttgcctttca gtcttcttga aatgctgtct ccctatctga 1441 aatccagctcacgtctgact tccaaaaccg tgcttgcctt taacttatgg aataaatatc 1501 tcaaacagatccccgggcga gctcgaattc gcggccgcac tcgagatatc tagacccagc 1561 tttcttgtacaaagtggtga tcgattcgac agatcactga aatgtgtggg cgtggcttaa 1621 gggtgggaaagaatatataa ggtgggggtc ttatgtagtt ttgtatctgt tttgcagcag 1681 ccgccgccgccatgagcacc aactcgtttg atggaagcat tgtgagctca tatttgacaa 1741 cgcgcatgcccccatgggcc ggggtgcgtc agaatgtgat gggctccagc attgatggtc 1801 gccccgtcctgcccgcaaac tctactacct tgacctacga gaccgtgtct ggaacgccgt 1861 tggagactgcagcctccgcc gccgcttcag ccgctgcagc caccgcccgc gggattgtga 1921 ctgactttgctttcctgagc ccgcttgcaa gcagtgcagc ttcccgttca tccgcccgcg 1981 atgacaagttgacggctctt ttggcacaat tggattcttt gacccgggaa cttaatgtcg 2041 tttctcagcagctgttggat ctgcgccagc aggtttctgc cctgaaggct tcctcccctc 2101 ccaatgcggtttaaaacata aataaaaaac cagactctgt ttggatttgg atcaagcaag 2161 tgtcttgctgtctttattta ggggttttgc gcgcgcggta ggcccgggac cagcggtctc 2221 ggtcgttgagggtcctgtgt attttttcca ggacgtggta aaggtgactc tggatgttca 2281 gatacatgggcataagcccg tctctggggt ggaggtagca ccactgcaga gcttcatgct 2341 gcggggtggtgttgtagatg atccagtcgt agcaggagcg ctgggcgtgg tgcctaaaaa 2401 tgtctttcagtagcaagctg attgccaggg gcaggccctt ggtgtaagtg tttacaaagc 2461 ggttaagctgggatgggtgc atacgtgggg atatgagatg catcttggac tgtattttta 2521 ggttggctatgttcccagcc atatccctcc ggggattcat gttgtgcaga accaccagca 2581 cagtgtatccggtgcacttg ggaaatttgt catgtagctt agaaggaaat gcgtggaaga 2641 acttggagacgcccttgtga cctccaagat tttccatgca ttcgtccata atgatggcaa 2701 tgggcccacgggcggcggcc tgggcgaaga tatttctggg atcactaacg tcatagttgt 2761 gttccaggatgagatcgtca taggccattt ttacaaagcg cgggcggagg gtgccagact 2821 gcggtataatggttccatcc ggcccagggg cgtagttacc ctcacagatt tgcatttccc 2881 acgctttgagttcagatggg gggatcatgt ctacctgcgg ggcgatgaag aaaacggttt 2941 ccggggtaggggagatcagc tgggaagaaa gcaggttcct gagcagctgc gacttaccgc 3001 agccggtgggcccgtaaatc acacctatta ccgggtgcaa ctggtagtta agagagctgc 3061 agctgccgtcatccctgagc aggggggcca cttcgttaag catgtccctg actcgcatgt 3121 tttccctgaccaaatccgcc agaaggcgct cgccgcccag cgatagcagt tcttgcaagg 3181 aagcaaagtttttcaacggt ttgagaccgt ccgccgtagg catgcttttg agcgtttgac 3241 caagcagttccaggcggtcc cacagctcgg tcacctgctc tacggcatct cgatccagca 3301 tatctcctcgtttcgcgggt tggggcggct ttcgctgtac ggcagtagtc ggtgctcgtc 3361 cagacgggccagggtcatgt ctttccacgg gcgcagggtc ctcgtcagcg tagtctgggt 3421 cacggtgaaggggtgcgctc cgggctgcgc gctggccagg gtgcgcttga ggctggtcct 3481 gctggtgctgaagcgctgcc ggtcttcgcc ctgcgcgtcg gccaggtagc atttgaccat 3541 ggtgtcatagtccagcccct ccgcggcgtg gcccttggcg cgcagcttgc ccttggagga 3601 ggcgccgcacgaggggcagt gcagactttt gagggcgtag agcttgggcg cgagaaatac 3661 cgattccggggagtaggcat ccgcgccgca ggccccgcag acggtctcgc attccacgag 3721 ccaggtgagctctggccgtt cggggtcaaa aaccaggttt cccccatgct ttttgatgcg 3781 tttcttacctctggtttcca tgagccggtg tccacgctcg gtgacgaaaa ggctgtccgt 3841 gtccccgtatacagacttga gaggcctgtc ctcgagcggt gttccgcggt cctcctcgta 3901 tagaaactcggaccactctg agacaaaggc tcgcgtccag gccagcacga aggaggctaa 3961 gtgggaggggtagcggtcgt tgtccactag ggggtccact cgctccaggg tgtgaagaca 4021 catgtcgccctcttcggcat caaggaaggt gattggtttg taggtgtagg ccacgtgacc 4081 gggtgttcctgaaggggggc tataaaaggg ggtgggggcg cgttcgtcct cactctcttc 4141 cgcatcgctgtctgcgaggg ccagctgttg gggtgagtac tccctctgaa aagcgggcat 4201 gacttctgcgctaagattgt cagtttccaa aaacgaggag gatttgatat tcacctggcc 4261 cgcggtgatgcctttgaggg tggccgcatc catctggtca gaaaagacaa tctttttgtt 4321 gtcaagcttggtggcaaacg acccgtagag ggcgttggac agcaacttgg cgatggagcg 4381 cagggtttggtttttgtcgc gatcggcgcg ctccttggcc gcgatgttta gctgcacgta 4441 ttcgcgcgcaacgcaccgcc attcgggaaa gacggtggtg cgctcgtcgg gcaccaggtg 4501 cacgcgccaaccgcggttgt gcagggtgac aaggtcaacg ctggtggcta cctctccgcg 4561 taggcgctcgttggtccagc agaggcggcc gcccttgcgc gagcagaatg gcggtagggg 4621 gtctagctgcgtctcgtccg gggggtctgc gtccacggta aagaccccgg gcagcaggcg 4681 cgcgtcgaagtagtctatct tgcatccttg caagtctagc gcctgctgcc atgcgcgggc 4741 ggcaagcgcgcgctcgtatg ggttgagtgg gggaccccat ggcatggggt gggtgagcgc 4801 ggaggcgtacatgccgcaaa tgtcgtaaac gtagaggggc tctctgagta ttccaagata 4861 tgtagggtagcatcttccac cgcggatgct ggcgcgcacg taatcgtata gttcgtgcga 4921 gggagcgaggaggtcgggac cgaggttgct acgggcgggc tgctctgctc ggaagactat 4981 ctgcctgaagatggcatgtg agttggatga tatggttgga cgctggaaga cgttgaagct 5041 ggcgtctgtgagacctaccg cgtcacgcac gaaggaggcg taggagtcgc gcagcttgtt 5101 gaccagctcggcggtgacct gcacgtctag ggcgcagtag tccagggttt ccttgatgat 5161 gtcatacttatcctgtccct tttttttcca cagctcgcgg ttgaggacaa actcttcgcg 5221 gtctttccagtactcttgga tcggaaaccc gtcggcctcc gaacggtaag agcctagcat 5281 gtagaactggttgacggcct ggtaggcgca gcatcccttt tctacgggta gcgcgtatgc 5341 ctgcgcggccttccggagcg aggtgtgggt gagcgcaaag gtgtccctga ccatgacttt 5401 gaggtactggtatttgaagt cagtgtcgtc gcatccgccc tgctcccaga gcaaaaagtc 5461 cgtgcgctttttggaacgcg gatttggcag ggcgaaggtg acatcgttga agagtatctt 5521 tcccgcgcgaggcataaagt tgcgtgtgat gcggaagggt cccggcacct cggaacggtt 5581 gttaattacctgggcggcga gcacgatctc gtcaaagccg ttgatgttgt ggcccacaat 5641 gtaaagttccaagaagcgcg ggatgccctt gatggaaggc aattttttaa gttcctcgta 5701 ggtgagctcttcaggggagc tgagcccgtg ctctgaaagg gcccagtctg caagatgagg 5761 gttggaagcgacgaatgagc tccacaggtc acgggccatt agcatttgca ggtggtcgcg 5821 aaaggtcctaaactggcgac ctatggccat tttttctggg gtgatgcagt agaaggtaag 5881 cgggtcttgttcccagcggt cccatccaag gttcgcggct aggtctcgcg cggcagtcac 5941 tagaggctcatctccgccga acttcatgac cagcatgaag ggcacgagct gcttcccaaa 6001 ggcccccatccaagtatagg tctctacatc gtaggtgaca aagagacgct cggtgcgagg 6061 atgcgagccgatcgggaaga actggatctc ccgccaccaa ttggaggagt ggctattgat 6121 gtggtgaaagtagaagtccc tgcgacgggc cgaacactcg tgctggcttt tgtaaaaacg 6181 tgcgcagtactggcagcggt gcacgggctg tacatcctgc acgaggttga cctgacgacc 6241 gcgcacaaggaagcagagtg ggaatttgag cccctcgcct ggcgggtttg gctggtggtc 6301 ttctacttcggctgcttgtc cttgaccgtc tggctgctcg aggggagtta cggtggatcg 6361 gaccaccacgccgcgcgagc ccaaagtcca gatgtccgcg cgcggcggtc ggagcttgat 6421 gacaacatcgcgcagatggg agctgtccat ggtctggagc tcccgcggcg tcaggtcagg 6481 cgggagctcctgcaggttta cctcgcatag acgggtcagg gcgcgggcta gatccaggtg 6541 atacctaatttccaggggct ggttggtggc ggcgtcgatg gcttgcaaga ggccgcatcc 6601 ccgcggcgcgactacggtac cgcgcggcgg gcggtgggcc gcgggggtgt ccttggatga 6661 tgcatctaaaagcggtgacg cgggcgagcc cccggaggta gggggggctc cggacccgcc 6721 gggagagggggcaggggcac gtcggcgccg cgcgcgggca ggagctggtg ctgcgcgcgt 6781 aggttgctggcgaacgcgac gacgcggcgg ttgatctcct gaatctggcg cctctgcgtg 6841 aagacgacgggcccggtgag cttgagcctg aaagagagtt cgacagaatc aatttcggtg 6901 tcgttgacggcggcctggcg caaaatctcc tgcacgtctc ctgagttgtc ttgataggcg 6961 atctcggccatgaactgctc gatctcttcc tcctggagat ctccgcgtcc ggctcgctcc 7021 acggtggcggcgaggtcgtt ggaaatgcgg gccatgagct gcgagaaggc gttgaggcct 7081 ccctcgttccagacgcggct gtagaccacg cccccttcgg catcgcgggc gcgcatgacc 7141 acctgcgcgagattgagctc cacgtgccgg gcgaagacgg cgtagtttcg caggcgctga 7201 aagaggtagttgagggtggt ggcggtgtgt tctgccacga agaagtacat aacccagcgt 7261 cgcaacgtggattcgttgat atcccccaag gcctcaaggc gctccatggc ctcgtagaag 7321 tccacggcgaagttgaaaaa ctgggagttg cgcgccgaca cggttaactc ctcctccaga 7381 agacggatgagctcggcgac agtgtcgcgc acctcgcgct caaaggctac aggggcctct 7441 tcttcttcttcaatctcctc ttccataagg gcctcccctt cttcttcttc tggcggcggt 7501 gggggaggggggacacggcg gcgacgacgg cgcaccggga ggcggtcgac aaagcgctcg 7561 atcatctccccgcggcgacg gcgcatggtc tcggtgacgg cgcggccgtt ctcgcggggg 7621 cgcagttggaagacgccgcc cgtcatgtcc cggttatggg ttggcggggg gctgccatgc 7681 ggcagggatacggcgctaac gatgcatctc aacaattgtt gtgtaggtac tccgccgccg 7741 agggacctgagcgagtccgc atcgaccgga tcggaaaacc tctcgagaaa ggcgtctaac 7801 cagtcacagtcgcaaggtag gctgagcacc gtggcgggcg gcagcgggcg gcggtcgggg 7861 ttgtttctggcggaggtgct gctgatgatg taattaaagt aggcggtctt gagacggcgg 7921 atggtcgacagaagcaccat gtccttgggt ccggcctgct gaatgcgcag gcggtcggcc 7981 atgccccaggcttcgttttg acatcggcgc aggtctttgt agtagtcttg catgagcctt 8041 tctaccggcacttcttcttc tccttcctct tgtcctgcat ctcttgcatc tatcgctgcg 8101 gcggcggcggagtttggccg taggtggcgc cctcttcctc ccatgcgtgt gaccccgaag 8161 cccctcatcggctgaagcag ggctaggtcg gcgacaacgc gctcggctaa tatggcctgc 8221 tgcacctgcgtgagggtaga ctggaagtca tccatgtcca caaagcggtg gtatgcgccc 8281 gtgttgatggtgtaagtgca gttggccata acggaccagt taacggtctg gtgacccggc 8341 tgcgagagctcggtgtacct gagacgcgag taagccctcg agtcaaatac gtagtcgttg 8401 caagtccgcaccaggtactg gtatcccacc aaaaagtgcg gcggcggctg gcggtagagg 8461 ggccagcgtagggtggccgg ggctccgggg gcgagatctt ccaacataag gcgatgatat 8521 ccgtagatgtacctggacat ccaggtgatg ccggcggcgg tggtggaggc gcgcggaaag 8581 tcgcggacgcggttccagat gttgcgcagc ggcaaaaagt gctccatggt cgggacgctc 8641 tggccggtcaggcgcgcgca atcgttgacg ctctagaccg tgcaaaagga gagcctgtaa 8701 gcgggcactcttccgtggtc tggtggataa attcgcaagg gtatcatggc ggacgaccgg 8761 ggttcgagccccgtatccgg ccgtccgccg tgatccatgc ggttaccgcc cgcgtgtcga 8821 acccaggtgtgcgacgtcag acaacggggg agtgctcctt ttggcttcct tccaggcgcg 8881 gcggctgctgcgctagcttt tttggccact ggccgcgcgc agcgtaagcg gttaggctgg 8941 aaagcgaaagcattaagtgg ctcgctccct gtagccggag ggttattttc caagggttga 9001 gtcgcgggacccccggttcg agtctcggac cggccggact gcggcgaacg ggggtttgcc 9061 tccccgtcatgcaagacccc gcttgcaaat tcctccggaa acagggacga gccccttttt 9121 tgcttttcccagatgcatcc ggtgctgcgg cagatgcgcc cccctcctca gcagcggcaa 9181 gagcaagagcagcggcagac atgcagggca ccctcccctc ctcctaccgc gtcaggaggg 9241 gcgacatccgcggttgacgc ggcagcagat ggtgattacg aacccccgcg gcgccgggcc 9301 cggcactacctggacttgga ggagggcgag ggcctggcgc ggctaggagc gccctctcct 9361 gagcggtacccaagggtgca gctgaagcgt gatacgcgtg aggcgtacgt gccgcggcag 9421 aacctgtttcgcgaccgcga gggagaggag cccgaggaga tgcgggatcg aaagttccac 9481 gcagggcgcgagctgcggca tggcctgaat cgcgagcggt tgctgcgcga ggaggacttt 9541 gagcccgacgcgcgaaccgg gattagtccc gcgcgcgcac acgtggcggc cgccgacctg 9601 gtaaccgcatacgagcagac ggtgaaccag gagattaact ttcaaaaaag ctttaacaac 9661 cacgtgcgtacgcttgtggc gcgcgaggag gtggctatag gactgatgca tctgtgggac 9721 tttgtaagcgcgctggagca aaacccaaat agcaagccgc tcatggcgca gctgttcctt 9781 atagtgcagcacagcaggga caacgaggca ttcagggatg cgctgctaaa catagtagag 9841 cccgagggccgctggctgct cgatttgata aacatcctgc agagcatagt ggtgcaggag 9901 cgcagcttgagcctggctga caaggtggcc gccatcaact attccatgct tagcctgggc 9961 aagttttacgcccgcaagat ataccatacc ccttacgttc ccatagacaa ggaggtaaag 10021 atcgaggggttctacatgcg catggcgctg aaggtgctta ccttgagcga cgacctgggc 10081 gtttatcgcaacgagcgcat ccacaaggcc gtgagcgtga gccggcggcg cgagctcagc 10141 gaccgcgagctgatgcacag cctgcaaagg gccctggctg gcacgggcag cggcgataga 10201 gaggccgagtcctactttga cgcgggcgct gacctgcgct gggccccaag ccgacgcgcc 10261 ctggaggcagctggggccgg acctgggctg gcggtggcac ccgcgcgcgc tggcaacgtc 10321 ggcggcgtggaggaatatga cgaggacgat gagtacgagc cagaggacgg cgagtactaa 10381 gcggtgatgtttctgatcag atgatgcaag acgcaacgga cccggcggtg cgggcggcgc 10441 tgcagagccagccgtccggc cttaactcca cggacgactg gcgccaggtc atggaccgca 10501 tcatgtcgctgactgcgcgc aatcctgacg cgttccggca gcagccgcag gccaaccggc 10561 tctccgcaattctggaagcg gtggtcccgg cgcgcgcaaa ccccacgcac gagaaggtgc 10621 tggcgatcgtaaacgcgctg gccgaaaaca gggccatccg gcccgacgag gccggcctgg 10681 tctacgacgcgctgcttcag cgcgtggctc gttacaacag cggcaacgtg cagaccaacc 10741 tggaccggctggtgggggat gtgcgcgagg ccgtggcgca gcgtgagcgc gcgcagcagc 10801 agggcaacctgggctccatg gttgcactaa acgccttcct gagtacacag cccgccaacg 10861 tgccgcggggacaggaggac tacaccaact ttgtgagcgc actgcggcta atggtgactg 10921 agacaccgcaaagtgaggtg taccagtctg ggccagacta ttttttccag accagtagac 10981 aaggcctgcagaccgtaaac ctgagccagg ctttcaaaaa cttgcagggg ctgtgggggg 11041 tgcgggctcccacaggcgac cgcgcgaccg tgtctagctt gctgacgccc aactcgcgcc 11101 tgttgctgctgctaatagcg cccttcacgg acagtggcag cgtgtcccgg gacacatacc 11161 taggtcacttgctgacactg taccgcgagg ccataggtca ggcgcatgtg gacgagcata 11221 ctttccaggagattacaagt gtcagccgcg cgctggggca ggaggacacg ggcagcctgg 11281 aggcaaccctaaactacctg ctgaccaacc ggcggcagaa gatcccctcg ttgcacagtt 11341 taaacagcgaggaggagcgc attttgcgct acgtgcagca gagcgtgagc cttaacctga 11401 tgcgcgacggggtaacgccc agcgtggcgc tggacatgac cgcgcgcaac atggaaccgg 11461 gcatgtatgcctcaaaccgg ccgtttatca accgcctaat ggactacttg catcgcgcgg 11521 ccgccgtgaaccccgagtat ttcaccaatg ccatcttgaa cccgcactgg ctaccgcccc 11581 ctggtttctacaccggggga ttcgaggtgc ccgagggtaa cgatggattc ctctgggacg 11641 acatagacgacagcgtgttt tccccgcaac cgcagaccct gctagagttg caacagcgcg 11701 agcaggcagaggcggcgctg cgaaaggaaa gcttccgcag gccaagcagc ttgtccgatc 11761 taggcgctgcggccccgcgg tcagatgcta gtagcccatt tccaagcttg atagggtctc 11821 ttaccagcactcgcaccacc cgcccgcgcc tgctgggcga ggaggagtac ctaaacaact 11881 cgctgctgcagccgcagcgc gaaaaaaacc tgcctccggc atttcccaac aacgggatag 11941 agagcctagtggacaagatg agtagatgga agacgtacgc gcaggagcac agggacgtgc 12001 caggcccgcgcccgcccacc cgtcgtcaaa ggcacgaccg tcagcggggt ctggtgtggg 12061 aggacgatgactcggcagac gacagcagcg tcctggattt gggagggagt ggcaacccgt 12121 ttgcgcaccttcgccccagg ctggggagaa tgttttaaaa aaaaaaaagc atgatgcaaa 12181 ataaaaaactcaccaaggcc atggcaccga gcgttggttt tcttgtattc cccttagtat 12241 gcggcgcgcggcgatgtatg aggaaggtcc tcctccctcc tacgagagtg tggtgagcgc 12301 ggcgccagtggcggcggcgc tgggttctcc cttcgatgct cccctggacc cgccgtttgt 12361 gcctccgcggtacctgcggc ctaccggggg gagaaacagc atccgttact ctgagttggc 12421 acccctattcgacaccaccc gtgtgtacct ggtggacaac aagtcaacgg atgtggcatc 12481 cctgaactaccagaacgacc acagcaactt tctgaccacg gtcattcaaa acaatgacta 12541 cagcccgggggaggcaagca cacagaccat caatcttgac gaccggtcgc actggggcgg 12601 cgacctgaaaaccatcctgc ataccaacat gccaaatgtg aacgagttca tgtttaccaa 12661 taagtttaaggcgcgggtga tggtgtcgcg cttgcctact aaggacaatc aggtggagct 12721 gaaatacgagtgggtggagt tcacgctgcc cgagggcaac tactccgaga ccatgaccat 12781 agaccttatgaacaacgcga tcgtggagca ctacttgaaa gtgggcagac agaacggggt 12841 tctggaaagcgacatcgggg taaagtttga cacccgcaac ttcagactgg ggtttgaccc 12901 cgtcactggtcttgtcatgc ctggggtata tacaaacgaa gccttccatc cagacatcat 12961 tttgctgccaggatgcgggg tggacttcac ccacagccgc ctgagcaact tgttgggcat 13021 ccgcaagcggcaacccttcc aggagggctt taggatcacc tacgatgatc tggagggtgg 13081 taacattcccgcactgttgg atgtggacgc ctaccaggcg agcttgaaag atgacaccga 13141 acagggcgggggtggcgcag gcggcagcaa cagcagtggc agcggcgcgg aagagaactc 13201 caacgcggcagccgcggcaa tgcagccggt ggaggacatg aacgatcatg ccattcgcgg 13261 cgacacctttgccacacggg ctgaggagaa gcgcgctgag gccgaagcag cggccgaagc 13321 tgccgcccccgctgcgcaac ccgaggtcga gaagcctcag aagaaaccgg tgatcaaacc 13381 cctgacagaggacagcaaga aacgcagtta caacctaata agcaatgaca gcaccttcac 13441 ccagtaccgcagctggtacc ttgcatacaa ctacggcgac cctcagaccg gaatccgctc 13501 atggaccctgctttgcactc ctgacgtaac ctgcggctcg gagcaggtct actggtcgtt 13561 gccagacatgatgcaagacc ccgtgacctt ccgctccacg cgccagatca gcaactttcc 13621 ggtggtgggcgccgagctgt tgcccgtgca ctccaagagc ttctacaacg accaggccgt 13681 ctactcccaactcatccgcc agtttacctc tctgacccac gtgttcaatc gctttcccga 13741 gaaccagattttggcgcgcc cgccagcccc caccatcacc accgtcagtg aaaacgttcc 13801 tgctctcacagatcacggga cgctaccgct gcgcaacagc atcggaggag tccagcgagt 13861 gaccattactgacgccagac gccgcacctg cccctacgtt tacaaggccc tgggcatagt 13921 ctcgccgcgcgtcctatcga gccgcacttt ttgagcaagc atgtccatcc ttatatcgcc 13981 cagcaataacacaggctggg gcctgcgctt cccaagcaag atgtttggcg gggccaagaa 14041 gcgctccgaccaacacccag tgcgcgtgcg cgggcactac cgcgcgccct ggggcgcgca 14101 caaacgcggccgcactgggc gcaccaccgt cgatgacgcc atcgacgcgg tggtggagga 14161 ggcgcgcaactacacgccca cgccgccacc agtgtccaca gtggacgcgg ccattcagac 14221 cgtggtgcgcggagcccggc gctatgctaa aatgaagaga cggcggaggc gcgtagcacg 14281 tcgccaccgccgccgacccg gcactgccgc ccaacgcgcg gcggcggccc tgcttaaccg 14341 cgcacgtcgcaccggccgac gggcggccat gcgggccgct cgaaggctgg ccgcgggtat 14401 tgtcactgtgccccccaggt ccaggcgacg agcggccgcc gcagcagccg cggccattag 14461 tgctatgactcagggtcgca ggggcaacgt gtattgggtg cgcgactcgg ttagcggcct 14521 gcgcgtgcccgtgcgcaccc gccccccgcg caactagatt gcaagaaaaa actacttaga 14581 ctcgtactgttgtatgtatc cagcggcggc ggcgcgcaac gaagctatgt ccaagcgcaa 14641 aatcaaagaagagatgctcc aggtcatcgc gccggagatc tatggccccc cgaagaagga 14701 agagcaggattacaagcccc gaaagctaaa gcgggtcaaa aagaaaaaga aagatgatga 14761 tgatgaacttgacgacgagg tggaactgct gcacgctacc gcgcccaggc gacgggtaca 14821 gtggaaaggtcgacgcgtaa aacgtgtttt gcgacccggc accaccgtag tctttacgcc 14881 cggtgagcgctccacccgca cctacaagcg cgtgtatgat gaggtgtacg gcgacgagga 14941 cctgcttgagcaggccaacg agcgcctcgg ggagtttgcc tacggaaagc ggcataagga 15001 catgctggcgttgccgctgg acgagggcaa cccaacacct agcctaaagc ccgtaacact 15061 gcagcaggtgctgcccgcgc ttgcaccgtc cgaagaaaag cgcggcctaa agcgcgagtc 15121 tggtgacttggcacccaccg tgcagctgat ggtacccaag cgccagcgac tggaagatgt 15181 cttggaaaaaatgaccgtgg aacctgggct ggagcccgag gtccgcgtgc ggccaatcaa 15241 gcaggtggcgccgggactgg gcgtgcagac cgtggacgtt cagataccca ctaccagtag 15301 caccagtattgccaccgcca cagagggcat ggagacacaa acgtccccgg ttgcctcagc 15361 ggtggcggatgccgcggtgc aggcggtcgc tgcggccgcg tccaagacct ctacggaggt 15421 gcaaacggacccgtggatgt ttcgcgtttc agccccccgg cgcccgcgcg gttcgaggaa 15481 gtacggcgccgccagcgcgc tactgcccga atatgcccta catccttcca ttgcgcctac 15541 ccccggctatcgtggctaca cctaccgccc cagaagacga gcaactaccc gacgccgaac 15601 caccactggaacccgccgcc gccgtcgccg tcgccagccc gtgctggccc cgatttccgt 15661 gcgcagggtggctcgcgaag gaggcaggac cctggtgctg ccaacagcgc gctaccaccc 15721 cagcatcgtttaaaagccgg tctttgtggt tcttgcagat atggccctca cctgccgcct 15781 ccgtttcccggtgccgggat tccgaggaag aatgcaccgt aggaggggca tggccggcca 15841 cggcctgacgggcggcatgc gtcgtgcgcac caccggcgg cggcgcgcgt cgcaccgtcg 15901 catgcgcggcggtatcctgc ccctccttat tccactgatc gccgcggcga ttggcgccgt 15961 gcccggaattgcatccgtgg ccttgcaggc gcagagacac tgattaaaaa caagttgcat 16021 gtggaaaaatcaaaataaaa agtctggact ctcacgctcg cttggtcctg taactatttt 16081 gtagaatggaagacatcaac tttgcgtctc tggccccgcg acacggctcg cgcccgttca 16141 tgggaaactggcaagatatc ggcaccagca atatgagcgg tggcgccttc agctggggct 16201 cgctgtggagcggcattaaa aatttcggtt ccaccgttaa gaactatggc agcaaggcct 16261 ggaacagcagcacaggccag atgctgaggg ataagttgaa agagcaaaat ttccaacaaa 16321 aggtggtagatggcctggcc tctggcatta gcggggtggt ggacctggcc aaccaggcag 16381 tgcaaaataagattaacagt aagcttgatc cccgccctcc cgtagaggag cctccaccgg 16441 ccgtggagacagtgtctcca gaggggcgtg gcgaaaagcg tccgcgcccc gacagggaag 16501 aaactctggtgacgcaaata gacgagcctc cctcgtacga ggaggcacta aagcaaggcc 16561 tgcccaccacccgtcccatc gcgcccatgg ctaccggagt gctgggccag cacacacccg 16621 taacgctggacctgcctccc cccgccgaca cccagcagaa acctgtgctg ccaggcccga 16681 ccgccgttgttgtaacccgt cctagccgcg cgtccctgcg ccgcgccgcc agcggtccgc 16741 gatcgttgcggcccgtagcc agtggcaact ggcaaagcac actgaacagc atcgtgggtc 16801 tgggggtgcaatccctgaag cgccgacgat gcttctgaat agctaacgtg tcgtatgtgt 16861 gtcatgtatgcgtccatgtc gccgccagag gagctgctga gccgccgcgc gcccgctttc 16921 caagatggctaccccttcga tgatgccgca gtggtcttac atgcacatct cgggccagga 16981 cgcctcggagtacctgagcc ccgggctggt gcagtttgcc cgcgccaccg agacgtactt 17041 cagcctgaataacaagttta gaaaccccac ggtggcgcct acgcacgacg tgaccacaga 17101 ccggtcccagcgtttgacgc tgcggttcat ccctgtggac cgtgaggata ctgcgtactc 17161 gtacaaggcgcggttcaccc tagctgtggg tgataaccgt gtgctggaca tggcttccac 17221 gtactttgacatccgcggcg tgctggacag gggccctact tttaagccct actctggcac 17281 tgcctacaacgccctggctc ccaagggtgc cccaaatcct tgcgaatggg atgaagctgc 17341 tactgctcttgaaataaacc tagaagaaga ggacgatgac aacgaagacg aagtagacga 17401 gcaagctgagcagcaaaaaa ctcacgtatt tgggcaggcg ccttattctg gtataaatat 17461 tacaaaggagggtattcaaa taggtgtcga aggtcaaaca cctaaatatg ccgataaaac 17521 atttcaacctgaacctcaaa taggagaatc tcagtggtac gaaactgaaa ttaatcatgc 17581 agctgggagagtccttaaaa agactacccc aatgaaacca tgttacggtt catatgcaaa 17641 acccacaaatgaaaatggag ggcaaggcat tcttgtaaag caacaaaatg gaaagctaga 17701 aagtcaagtggaaatgcaat ttttctcaac tactgaggcg accgcaggca atggtgataa 17761 cttgactcctaaagtggtat tgtacagtga agatgtagat atagaaaccc cagacactca 17821 tatttcttacatgcccacta ttaaggaagg taactcacga gaactaatgg gccaacaatc 17881 tatgcccaacaggcctaatt acattgcttt tagggacaat tttattggtc taatgtatta 17941 caacagcacgggtaatatgg gtgttctggc gggccaagca tcgcagttga atgctgttgt 18001 agatttgcaagacagaaaca cagagctttc ataccagctt ttgcttgatt ccattggtga 18061 tagaaccaggtacttttcta tgtggaatca ggctgttgac agctatgatc cagatgttag 18121 aattattgaaaatcatggaa ctgaagatga acttccaaat tactgctttc cactgggagg 18181 tgtgattaatacagagactc ttaccaaggt aaaacctaaa acaggtcagg aaaatggatg 18241 ggaaaaagatgctacagaat tttcagataa aaatgaaata agagttggaa ataattttgc 18301 catggaaatcaatctaaatg ccaacctgtg gagaaatttc ctgtactcca acatagcgct 18361 gtatttgcccgacaagctaa agtacagtcc ttccaacgta aaaatttctg ataacccaaa 18421 cacctacgactacatgaaca agcgagtggt ggctcccggg ttagtggact gctacattaa 18481 ccttggagcacgctggtccc ttgactatat ggacaacgtc aacccattta accaccaccg 18541 caatgctggcctgcgctacc gctcaatgtt gctgggcaat ggtcgctatg tgcccttcca 18601 catccaggtgcctcagaagt tctttgccat taaaaacctc cttctcctgc cgggctcata 18661 cacctacgagtggaacttca ggaaggatgt taacatggtt ctgcagagct ccctaggaaa 18721 tgacctaagggttgacggag ccagcattaa gtttgatagc atttgccttt acgccacctt 18781 cttccccatggcccacaaca ccgcctccac gcttgaggcc atgcttagaa acgacaccaa 18841 cgaccagtcctttaacgact atctctccgc cgccaacatg ctctacccta tacccgccaa 18901 cgctaccaacgtgcccatat ccatcccctc ccgcaactgg gcggctttcc gcggctgggc 18961 cttcacgcgccttaagacta aggaaacccc atcactgggc tcgggctacg acccttatta 19021 cacctactctggctctatac cctacctaga tggaaccttt tacctcaacc acacctttaa 19081 gaaggtggccattacctttg actcttctgt cagctggcct ggcaatgacc gcctgcttac 19141 ccccaacgagtttgaaatta agcgctcagt tgacggggag ggttacaacg ttgcccagtg 19201 taacatgaccaaagactggt tcctggtaca aatgctagct aactacaaca ttggctacca 19261 gggcttctatatcccagaga gctacaagga ccgcatgtac tccttcttta gaaacttcca 19321 gcccatgagccgtcaggtgg tggatgatac taaatacaag gactaccaac aggtgggcat 19381 cctacaccaacacaacaact ctggatttgt tggctacctt gcccccacca tgcgcgaagg 19441 acaggcctaccctgctaact tcccctatcc gcttataggc aagaccgcag ttgacagcat 19501 tacccagaaaaagtttcttt gcgatcgcac cctttggcgc atcccattct ccagtaactt 19561 tatgtccatgggcgcactca cagacctggg ccaaaacctt ctctacgcca actccgccca 19621 cgcgctagacatgacttttg aggtggatcc catggacgag cccacccttc tttatgtttt 19681 gtttgaagtctttgacgtgg tccgtgtgca ccggccgcac cgcggcgtca tcgaaaccgt 19741 gtacctgcgcacgcccttct cggccggcaa cgccacaaca taaagaagca agcaacatca 19801 acaacagctgccgccatggg ctccagtgag caggaactga aagccattgt caaagatctt 19861 ggttgtgggccatatttttt gggcacctat gacaagcgct ttccaggctt tgtttctcca 19921 cacaagctcgcctgcgccat agtcaatacg gccggtcgcg agactggggg cgtacactgg 19981 atggcctttgcctggaaccc gcactcaaaa acatgctacc tctttgagcc ctttggcttt 20041 tctgaccagcgactcaagca ggtttaccag tttgagtacg agtcactcct gcgccgtagc 20101 gccattgcttcttcccccga ccgctgtata acgctggaaa agtccaccca aagcgtacag 20161 gggcccaactcggccgcctg tggactattc tgctgcatgt ttctccacgc ctttgccaac 20221 tggccccaaactcccatgga tcacaacccc accatgaacc ttattaccgg ggtacccaac 20281 tccatgctcaacagtcccca ggtacagccc accctgcgtc gcaaccagga acagctctac 20341 agcttcctggagcgccactc gccctacttc cgcagccaca gtgcgcagat taggagcgcc 20401 acttctttttgtcacttgaa aaacatgtaa aaataatgta ctagagacac tttcaataaa 20461 ggcaaatgcttttatttgta cactctcggg tgattattta cccccaccct tgccgtctgc 20521 gccgtttaaaaatcaaaggg gttctgccgc gcatcgctat gcgccactgg cagggacacg 20581 ttgcgatactggtgtttagt gctccactta aactcaggca caaccatccg cggcagctcg 20641 gtgaagttttcactccacag gctgcgcacc atcaccaacg cgtttagcag gtcgggcgcc 20701 gatatcttgaagtcgcagtt ggggcctccg ccctgcgcgc gcgagttgcg atacacaggg 20761 ttgcagcactggaacactat cagcgccggg tggtgcacgc tggccagcac gctcttgtcg 20821 gagatcagatccgcgtccag gtcctccgcg ttgctcaggg cgaacggagt caactttggt 20881 agctgccttcccaaaaaggg cgcgtgccca ggctttgagt tgcactcgca ccgtagtggc 20941 atcaaaaggtgaccgtgccc ggtctgggcg ttaggataca gcgcctgcat aaaagccttg 21001 atctgcttaaaagccacctg agcctttgcg ccttcagaga agaacatgcc gcaagacttg 21061 ccggaaaactgattggccgg acaggccgcg tcgtgcacgc agcaccttgc gtcggtgttg 21121 gagatctgcaccacatttcg gccccaccgg ttcttcacga tcttggcctt gctagactgc 21181 tccttcagcgcgcgctgccc gttttcgctc gtcacatcca tttcaatcac gtgctcctta 21241 tttatcataatgcttccgtg tagacactta agctcgcctt cgatctcagc gcagcggtgc 21301 agccacaacgcgcagcccgt gggctcgtga tgcttgtagg tcacctctgc aaacgactgc 21361 aggtacgcctgcaggaatcg ccccatcatc gtcacaaagg tcttgttgct ggtgaaggtc 21421 agctgcaacccgcggtgctc ctcgttcagc caggtcttgc atacggccgc cagagcttcc 21481 acttggtcaggcagtagttt gaagttcgcc tttagatcgt tatccacgtg gtacttgtcc 21541 atcagcgcgcgcgcagcctc catgcccttc tcccacgcag acacgatcgg cacactcagc 21601 gggttcatcaccgtaatttc actttccgct tcgctgggct cttcctcttc ctcttgcgtc 21661 cgcataccacgcgccactgg gtcgtcttca ttcagccgcc gcactgtgcg cttacctcct 21721 ttgccatgcttgattagcac cggtgggttg ctgaaaccca ccatttgtag cgccacatct 21781 tctctttcttcctcgctgtc cacgattacc tctggtgatg gcgggcgctc gggcttggga 21841 gaagggcgcttctttttctt cttgggcgca atggccaaat ccgccgccga ggtcgatggc 21901 cgcgggctgggtgtgcgcgg caccagcgcg tcttgtgatg agtcttcctc gtcctcggac 21961 tcgatacgccgcctcatccg cttttttggg ggcgcccggg gaggcggcgg cgacggggac 22021 ggggacgacacgtcctccat ggttggggga cgtcgcgccg caccgcgtcc gcgctcgggg 22081 gtggtttcgcgctgctcctc ttcccgactg gccatttcct tctcctatag gcagaaaaag 22141 atcatggagtcagtcgagaa gaaggacagc ctaaccgccc cctctgagtt cgccaccacc 22201 gcctccaccgatgccgccaa cgcgcctacc accttccccg tcgaggcacc cccgcttgag 22261 gaggaggaagtgattatcga gcaggaccca ggttttgtaa gcgaagacga cgaggaccgc 22321 tcagtaccaacagaggataa aaagcaagac caggacaacg cagaggcaaa cgaggaacaa 22381 gtcgggcggggggacgaaag gcatggcgac tacctagatg tgggagacga cgtgctgttg 22441 aagcatctgcagcgccagtg cgccattatc tgcgacgcgt tgcaagagcg cagcgatgtg 22501 cccctcgccatagcggatgt cagccttgcc tacgaacgcc acctattctc accgcgcgta 22561 ccccccaaacgccaagaaaa cggcacatgc gagcccaacc cgcgcctcaa cttctacccc 22621 gtatttgccgtgccagaggt gcttgccacc tatcacatct ttttccaaaa ctgcaagata 22681 cccctatcctgccgtgccaa ccgcagccga gcggacaagc agctggcctt gcggcagggc 22741 gctgtcatacctgatatcgc ctcgctcaac gaagtgccaa aaatctttga gggtcttgga 22801 cgcgacgagaagcgcgcggc aaacgctctg caacaggaaa acagcgaaaa tgaaagtcac 22861 tctggagtgttggtggaact cgagggtgac aacgcgcgcc tagccgtact aaaacgcagc 22921 atcgaggtcacccactttgc ctacccggca cttaacctac cccccaaggt catgagcaca 22981 gtcatgagtgagctgatcgt gcgccgtgcg cagcccctgg agagggatgc aaatttgcaa 23041 gaacaaacagaggagggcct acccgcagtt ggcgacgagc agctagcgcg ctggcttcaa 23101 acgcgcgagcctgccgactt ggaggagcga cgcaaactaa tgatggccgc agtgctcgtt 23161 accgtggagcttgagtgcat gcagcggttc tttgctgacc cggagatgca gcgcaagcta 23221 gaggaaacattgcactacac ctttcgacag ggctacgtac gccaggcctg caagatctcc 23281 aacgtggagctctgcaacct ggtctcctac cttggaattt tgcacgaaaa ccgccttggg 23341 caaaacgtgcttcattccac gctcaagggc gaggcgcgcc gcgactacgt ccgcgactgc 23401 gtttacttatttctatgcta cacctggcag acggccatgg gcgtttggca gcagtgcttg 23461 gaggagtgcaacctcaagga gctgcagaaa ctgctaaagc aaaacttgaa ggacctatgg 23521 acggccttcaacgagcgctc cgtggccgcg cacctggcgg acatcatttt ccccgaacgc 23581 ctgcttaaaaccctgcaaca gggtctgcca gacttcacca gtcaaagcat gttgcagaac 23641 tttaggaactttatcctaga gcgctcagga atcttgcccg ccacctgctg tgcacttcct 23701 agcgactttgtgcccattaa gtaccgcgaa tgccctccgc cgctttgggg ccactgctac 23761 cttctgcagctagccaacta ccttgcctac cactctgaca taatggaaga cgtgagcggt 23821 gacggtctactggagtgtca ctgtcgctgc aacctatgca ccccgcaccg ctccctggtt 23881 tgcaattcgcagctgcttaa cgaaagtcaa attatcggta cctttgagct gcagggtccc 23941 tcgcctgacgaaaagtccgc ggctccgggg ttgaaactca ctccggggct gtggacgtcg 24001 gcttaccttcgcaaatttgt acctgaggac taccacgccc acgagattag gttctacgaa 24061 gaccaatcccgcccgccaaa tgcggagctt accgcctgcg tcattaccca gggccacatt 24121 cttggccaattgcaagccat caacaaagcc cgccaagagt ttctgctacg aaagggacgg 24181 ggggtttacttggaccccca gtccggcgag gagctcaacc caatcccccc gccgccgcag 24241 ccctatcagcagcagccgcg ggcccttgct tcccaggatg gcacccaaaa agaagctgca 24301 gctgccgccgccacccacgg acgaggagga atactgggac agtcaggcag aggaggtttt 24361 ggacgaggaggaggaggaca tgatggaaga ctgggagagc ctagacgagg aagcttccga 24421 ggtcgaagaggtgtcagacg aaacaccgtc accctcggtc gcattcccct cgccggcgcc 24481 ccagaaatcggcaaccggtt ccagcatggc tacaacctcc gctcctcagg cgccgccggc 24541 actgcccgttcgccgaccca accgtagatg ggacaccact ggaaccaggg ccggtaagtc 24601 caagcagccgccgccgttag cccaagagca acaacagcgc caaggctacc gctcatggcg 24661 cgggcacaagaacgccatag ttgcttgctt gcaagactgt gggggcaaca tctccttcgc 24721 ccgccgctttcttctctacc atcacggcgt ggccttcccc cgtaacatcc tgcattacta 24781 ccgtcatctctacagcccat actgcaccgg cggcagcggc agcggcagca acagcagcgg 24841 ccacacagaagcaaaggcga ccggatagca agactctgac aaagcccaag aaatccacag 24901 cggcggcagcagcaggagga ggagcgctgc gtctggcgcc caacgaaccc gtatcgaccc 24961 gcgagcttagaaacaggatt tttcccactc tgtatgctat atttcaacag agcaggggcc 25021 aagaacaagagctgaaaata aaaaacaggt ctctgcgatc cctcacccgc agctgcctgt 25081 atcacaaaagcgaagatcag cttcggcgca cgctggaaga cgcggaggct ctcttcagta 25141 aatactgcgcgctgactctt aaggactagt ttcgcgccct ttctcaaatt taagcgcgaa 25201 aactacgtcatctccagcgg ccacacccgg cgccagcacc tgtcgtcagc gccattatga 25261 gcaaggaaattcccacgccc tacatgtgga gttaccagcc acaaatggga cttgcggctg 25321 gagctgcccaagactactca acccgaataa actacatgag cgcgggaccc cacatgatat 25381 cccgggtcaacggaatccgc gcccaccgaa accgaattct cttggaacag gcggctatta 25441 ccaccacacctcgtaataac cttaatcccc gtagttggcc cgctgccctg gtgtaccagg 25501 aaagtcccgctcccaccact gtggtacttc ccagagacgc ccaggccgaa gttcagatga 25561 ctaactcaggggcgcagctt gcgggcggct ttcgtcacag ggtgcggtcg cccgggcagg 25621 gtataactcacctgacaatc agagggcgag gtattcagct caacgacgag tcggtgagct 25681 cctcgcttggtctccgtccg gacgggacat ttcagatcgg cggcgccggc cgtccttcat 25741 tcacgcctcgtcaggcaatc ctaactctgc agacctcgtc ctctgagccg cgctctggag 25801 gcattggaactctgcaattt attgaggagt ttgtgccatc ggtctacttt aaccccttct 25861 cgggacctcccggccactat ccggatcaat ttattcctaa ctttgacgcg gtaaaggact 25921 cggcggacggctacgactga atgttaagtg gagaggcaga gcaactgcgc ctgaaacacc 25981 tggtccactgtcgccgccac aagtgctttg cccgcgactc cggtgagttt tgctactttg 26041 aattgcccgaggatcatatc gagggcccgg cgcacggcgt ccggcttacc gcccagggag 26101 agcttgcccgtagcctgatt cgggagttta cccagcgccc cctgctagtt gagcgggaca 26161 ggggaccctgtgttctcact gtgatttgca actgtcctaa ccttggatta catcaagatc 26221 tttgttgccatctctgtgct gagtataata aatacagaaa ttaaaatata ctggggctcc 26281 tatcgccatcctgtaaacgc caccgtcttc acccgcccaa gcaaaccaag gcgaacctta 26341 cctggtacttttaacatctc tccctctgtg atttacaaca gtttcaaccc agacggagtg 26401 agtctacgagagaacctctc cgagctcagc tactccatca gaaaaaacac caccctcctt 26461 acctgccgggaacgtacgag tgcgtcaccg gccgctgcac cacacctacc gcctgaccgt 26521 aaaccagactttttccggac agacctcaat aactctgttt accagaacag gaggtgagct 26581 tagaaaacccttagggtatt aggccaaagg cgcagctact gtggggttta tgaacaattc 26641 aagcaactctacgggctatt ctaattcagg tttctctaga aatggacgga attattacag 26701 agcagcgcctgctagaaaga cgcagggcag cggccgagca acagcgcatg aatcaagagc 26761 tccaagacatggttaacttg caccagtgca aaaggggtat cttttgtctg gtaaagcagg 26821 ccaaagtcacctacgacagt aataccaccg gacaccgcct tagctacaag ttgccaacca 26881 agcgtcagaaattggtggtc atggtgggag aaaagcccat taccataact cagcactcgg 26941 tagaaaccgaaggctgcatt cactcacctt gtcaaggacc tgaggatctc tgcaccctta 27001 ttaagaccctgtgcggtctc aaagatctta ttccctttaa ctaataaaaa aaaataataa 27061 agcatcacttacttaaaatc agttagcaaa tttctgtcca gtttattcag cagcacctcc 27121 ttgccctcctcccagctctg gtattgcagc ttcctcctgg ctgcaaactt tctccacaat 27181 ctaaatggaatgtcagtttc ctcctgttcc tgtccatccg cacccactat cttcatgttg 27241 ttgcagatgaagcgcgcaag accgtctgaa gataccttca accccgtgta tccatatgac 27301 acggaaaccggtcctccaac tgtgcctttt cttactcctc cctttgtatc ccccaatggg 27361 tttcaagagagtccccctgg ggtactctct ttgcgcctat ccgaacctct agttacctcc 27421 aatggcatgcttgcgctcaa aatgggcaac ggcctctctc tggacgaggc cggcaacctt 27481 acctcccaaaatgtaaccac tgtgagccca cctctcaaaa aaaccaagtc aaacataaac 27541 ctggaaatatctgcacccct cacagttacc tcagaagccc taactgtggc tgccgccgca 27601 cctctaatggtcgcgggcaa cacactcacc atgcaatcac aggccccgct aaccgtgcac 27661 gactccaaacttagcattgc cacccaagga cccctcacag tgtcagaagg aaagctagcc 27721 ctgcaaacatcaggccccct caccaccacc gatagcagta cccttactat cactgcctca 27781 ccccctctaactactgccac tggtagcttg ggcattgact tgaaagagcc catttataca 27841 caaaatggaaaactaggact aaagtacggg gctcctttgc atgtaacaga cgacctaaac 27901 actttgaccgtagcaactgg tccaggtgtg actattaata atacttcctt gcaaactaaa 27961 gttactggagccttgggttt tgattcacaa ggcaatatgc aacttaatgt agcaggagga 28021 ctaaggattgattctcaaaa cagacgcctt atacttgatg ttagttatcc gtttgatgct 28081 caaaaccaactaaatctaag actaggacag ggccctcttt ttataaactc agcccacaac 28141 ttggatattaactacaacaa aggcctttac ttgtttacag cttcaaacaa ttccaaaaag 28201 cttgaggttaacctaagcac tgccaagggg ttgatgtttg acgctacagc catagccatt 28261 aatgcaggagatgggcttga atttggttca cctaatgcac caaacacaaa tcccctcaaa 28321 acaaaaattggccatggcct agaatttgat tcaaacaagg ctatggttcc taaactagga 28381 actggccttagttttgacag cacaggtgcc attacagtag gaaacaaaaa taatgataag 28441 ctaactttgtggaccacacc agctccatct cctaactgta gactaaatgc agagaaagat 28501 gctaaactcactttggtctt aacaaaatgt ggcagtcaaa tacttgctac agtttcagtt 28561 ttggctgttaaaggcagttt ggctccaata tctggaacag ttcaaagtgc tcatcttatt 28621 ataagatttgacgaaaatgg agtgctacta aacaattcct tcctggaccc agaatattgg 28681 aactttagaaatggagatct tactgaaggc acagcctata caaacgctgt tggatttatg 28741 cctaacctatcagcttatcc aaaatctcac ggtaaaactg ccaaaagtaa cattgtcagt 28801 caagtttacttaaacggaga caaaactaaa cctgtaacac taaccattac actaaacggt 28861 acacaggaaacaggagacac aactccaagt gcatactcta tgtcattttc atgggactgg 28921 tctggccacaactacattaa tgaaatattt gccacatcct cttacacttt ttcatacatt 28981 gcccaagaataaagaatcgt ttgtgttatg tttcaacgtg tttatttttc aattgcagaa 29041 aatttcgaatcatttttcat tcagtagtat agccccacca ccacatagct tatacagatc 29101 accgtaccttaatcaaactc acagaaccct agtattcaac ctgccacctc cctcccaaca 29161 cacagagtacacagtccttt ctccccggct ggccttaaaa agcatcatat catgggtaac 29221 agacatattcttaggtgtta tattccacac ggtttcctgt cgagccaaac gctcatcagt 29281 gatattaataaactccccgg gcagctcact taagttcatg tcgctgtcca gctgctgagc 29341 cacaggctgctgtccaactt gcggttgctt aacgggcggc gaaggagaag tccacgccta 29401 catgggggtagagtcataat cgtgcatcag gatagggcgg tggtgctgca gcagcgcgcg 29461 aataaactgctgccgccgcc gctccgtcct gcaggaatac aacatggcag tggtctcctc 29521 agcgatgattcgcaccgccc gcagcataag gcgccttgtc ctccgggcac agcagcgcac 29581 cctgatctcacttaaatcag cacagtaact gcagcacagc accacaatat tgttcaaaat 29641 cccacagtgcaaggcgctgt atccaaagct catggcgggg accacagaac ccacgtggcc 29701 atcataccacaagcgcaggt agattaagtg gcgacccctc ataaacacgc tggacataaa 29761 cattacctcttttggcatgt tgtaattcac cacctcccgg taccatataa acctctgatt 29821 aaacatggcgccatccacca ccatcctaaa ccagctggcc aaaacctgcc cgccggctat 29881 acactgcagggaaccgggac tggaacaatg acagtggaga gcccaggact cgtaaccatg 29941 gatcatcatgctcgtcatga tatcaatgtt ggcacaacac aggcacacgt gcatacactt 30001 cctcaggattacaagctcct cccgcgttag aaccatatcc cagggaacaa cccattcctg 30061 aatcagcgtaaatcccacac tgcagggaag acctcgcacg taactcacgt tgtgcattgt 30121 caaagtgttacattcgggca gcagcggatg atcctccagt atggtagcgc gggtttctgt 30181 ctcaaaaggaggtagacgat ccctactgta cggagtgcgc cgagacaacc gagatcgtgt 30241 tggtcgtagtgtcatgccaa atggaacgcc ggacgtagtc atatttcctg aagcaaaacc 30301 aggtgcgggcgtgacaaaca gatctgcgtc tccggtctcg ccgcttagat cgctctgtgt 30361 agtagttgtagtatatccac tctctcaaag catccaggcg ccccctggct tcgggttcta 30421 tgtaaactccttcatgcgcc gctgccctga taacatccac caccgcagaa taagccacac 30481 ccagccaacctacacattcg ttctgcgagt cacacacggg aggagcggga agagctggaa 30541 gaaccatgttttttttttta ttccaaaaga ttatccaaaa cctcaaaatg aagatctatt 30601 aagtgaacgcgctcccctcc ggtggcgtgg tcaaactcta cagccaaaga acagataatg 30661 gcatttgtaagatgttgcac aatggcttcc aaaaggcaaa cggccctcac gtccaagtgg 30721 acgtaaaggctaaacccttc agggtgaatc tcctctataa acattccagc accttcaacc 30781 atgcccaaataattctcatc tcgccacctt ctcaatatat ctctaagcaa atcccgaata 30841 ttaagtccggccattgtaaa aatctgctcc agagcgccct ccaccttcag cctcaagcag 30901 cgaatcatgattgcaaaaat tcaggttcct cacagacctg tataagattc aaaagcggaa 30961 cattaacaaaaataccgcga tcccgtaggt cccttcgcag ggccagctga acataatcgt 31021 gcaggtctgcacggaccagc gcggccactt ccccgccagg aaccttgaca aaagaaccca 31081 cactgattatgacacgcata ctcggagcta tgctaaccag cgtagccccg atgtaagctt 31141 tgttgcatgggcggcgatat aaaatgcaag gtgctgctca aaaaatcagg caaagcctcg 31201 cgcaaaaaagaaagcacatc gtagtcatgc tcatgcagat aaaggcaggt aagctccgga 31261 accaccacagaaaaagacac catttttctc tcaaacatgt ctgcgggttt ctgcataaac 31321 acaaaataaaataacaaaaa aacatttaaa cattagaagc ctgtcttaca acaggaaaaa 31381 caacccttataagcataaga cggactacgg ccatgccggc gtgaccgtaa aaaaactggt 31441 caccgtgattaaaaagcacc accgacagct cctcggtcat gtccggagtc ataatgtaag 31501 actcggtaaacacatcaggt tgattcacat cggtcagtgc taaaaagcga ccgaaatagc 31561 ccgggggaatacatacccgc aggcgtagag acaacattac agcccccata ggaggtataa 31621 caaaattaataggagagaaa aacacataaa cacctgaaaa accctcctgc ctaggcaaaa 31681 tagcaccctcccgctccaga acaacataca gcgcttccac agcggcagcc ataacagtca 31741 gccttaccagtaaaaaagaa aacctattaa aaaaacacca ctcgacacgg caccagctca 31801 atcagtcacagtgtaaaaaa gggccaagtg cagagcgagt atatatagga ctaaaaaatg 31861 acgtaacggttaaagtccac aaaaaacacc cagaaaaccg cacgcgaacc tacgcccaga 31921 aacgaaagccaaaaaaccca caacttcctc aaatcgtcac ttccgttttc ccacgttacg 31981 tcacttcccattttaagaaa actacaattc ccaacacata caagttactc cgccctaaaa 32041 cctacgtcacccgccccgtt cccacgcccc gcgccacgtc acaaactcca ccccctcatt 32101 atcatattggcttcaatcca aaataaggta tattattgat gatgttaatt aatttaaatc 32161 cgcatgcgatatcgagctct cccgggaatt cggatctgcg acgcgaggct ggatggcctt 32221 ccccattatgattcttctcg cttccggcgg catcgggatg cccgcgttgc aggccatgct 32281 gtccaggcaggtagatgacg accatcaggg acagcttcac ggccagcaaa aggccaggaa 32341 ccgtaaaaaggccgcgttgc tggcgttttt ccataggctc cgcccccctg acgagcatca 32401 caaaaatcgacgctcaagtc agaggtggcg aaacccgaca ggactataaa gataccaggc 32461 gtttccccctggaagctccc tcgtgcgctc tcctgttccg accctgccgc ttaccggata 32521 cctgtccgcctttctccctt cgggaagcgt ggcgctttct caatgctcac gctgtaggta 32581 tctcagttcggtgtaggtcg ttcgctccaa gctgggctgt gtgcacgaac cccccgttca 32641 gcccgaccgctgcgccttat ccggtaacta tcgtcttgag tccaacccgg taagacacga 32701 cttatcgccactggcagcag ccactggtaa caggattagc agagcgaggt atgtaggcgg 32761 tgctacagagttcttgaagt ggtggcctaa ctacggctac actagaagga cagtatttgg 32821 tatctgcgctctgctgaagc cagttacctt cggaaaaaga gttggtagct cttgatccgg 32881 caaacaaaccaccgctggta gcggtggttt ttttgtttgc aagcagcaga ttacgcgcag 32941 aaaaaaaggatctcaagaag atcctttgat cttttctacg gggtctgacg ctcagtggaa 33001 cgaaaactcacgttaaggga ttttggtcat gagattatca aaaaggatct tcacctagat 33061 ccttttaaatcaatctaaag tatatatgag taaacttggt ctgacagtta ccaatgctta 33121 atcagtgaggcacctatctc agcgatctgt ctatttcgtt catccatagt tgcctgactc 33181 cccgtcgtgtagataactac gatacgggag ggcttaccat ctggccccag tgctgcaatg 33241 ataccgcgagacccacgctc accggctcca gatttatcag caataaacca gccagccgga 33301 agggccgagcgcagaagtgg tcctgcaact ttatccgcct ccatccagtc tattaattgt 33361 tgccgggaagctagagtaag tagttcgcca gttaatagtt tgcgcaacgt tgttgccatt 33421 gntgcaggcatcgtggtgtc acgctcgtcg tttggtatgg cttcattcag ctccggttcc 33481 caacgatcaaggcgagttac atgatccccc atgttgtgca aaaaagcggt tagctccttc 33541 ggtcctccgatcgttgtcag aagtaagttg gccgcagtgt tatcactcat ggttatggca 33601 gcactgcataattctcttac tgtcatgcca tccgtaagat gcttttctgt gactggtgag 33661 tactcaaccaagtcattctg agaatagtgt atgcggcgac cgagttgctc ttgcccggcg 33721 tcaacacgggataataccgc gccacatagc agaactttaa aagtgctcat cattggaaaa 33781 cgttcttcggggcgaaaact ctcaaggatc ttaccgctgt tgagatccag ttcgatgtaa 33841 cccactcgtgcacccaactg atcttcagca tcttttactt tcaccagcgt ttctgggtga 33901 gcaaaaacaggaaggcaaaa tgccgcaaaa aagggaataa gggcgacacg gaaatgttga 33961 atactcatactcttcctttt tcaatattat tgaagcattt atcagggtta ttgtctcatg 34021 agcggatacatatttgaatg tatttagaaa aataaacaaa taggggttcc gcgcacattt 34081 ccccgaaaagtgccacctga cgtctaagaa accattatta tcatgacatt aacctataaa 34141 aataggcgtatcacgaggcc ctttcgtctt caaggatccg aattcccggg agagctcgat 34201 atcgcatgcggatttaaatt aattaa

[1241] TABLE 9 Nucleotide sequence of a Sau3A fragment used to constructvectors comprising suppressor tRNA sequences. 1 ctagaggatc gaaaccatcctctgctatat ggccgcatat attttacttg aagactagga 61 ccctacagaa aaggggttttaaagtaggcg tgctaaacgt cagcggacct gacccgtgta 121 agaatccaca aggtatcctggtggaaatgc gcatttgtag gcttcaatat ctgtaatcct 181 actaattagg tgtggagagctttcagccag tttcgtaggt ttggagacca tttaggggtt 241 ggcgtgtggc cccctcgtaaagtctttcgt acttcctaca tcagacaagt cttgcaattt 301 gcaatatctc ttttagccaatatctaaatc tttaaaattt tgattttgtt ttttaaccag 361 gatgagagac attccagagttgttaccttg tcaaaataaa caaatttaaa gatgtctgtg 421 aaaagaaaca tatattcctcatgggaatat atccaggttg ttgaaggagg tacactcgag 481 tctccctatc agtgatagagatctcgaggt cgtagtcgtg gccgagtggt taaggcgatg 541 gactctaaat ccattggggtctccccgcgc aggttcgaat cctgccgact acggcgtgct 601 ttttttactc tcgggtagaggaaatccggt gcactacctg tgcaatcaca cagaataaca 661 tggagtagta ctttttattttcctgttatt atctttctcc ataaaagtgg aaccagataa 721 ttttagttct tttgtgtaacaagactagag attttttgaa gtgttacatt ggaaagcact 781 tgaaaacaca agtaatttctgacactgcta taaaaatgat ggaaaaacgc tcaagttgtt 841 ttgcctttca gtcttcttgaaatgctgtct ccctatctga aatccagctc acgtctgact 901 tccaaaaccg tgcttgcctttaacttatgg aataaatatc tcaaacagat cccc

[1242] TABLE 10 Nucleotide sequence of pAd/PL-DEST ™.CATCATCAATAATATACCTTATTTTGGATTGAAGCCAATATGATAATGAGGGGGTGGAGTTTGTGACGTGGCGCGGGGCGTGGGAACGGGGCGGGTGACGTAGTAGTGTGGCGGAAGTGTGATGTTGCAAGTGTGGCGGAACACATGTAAGCGACGGATGTGGCAAAAGTGACGTTTTTGGTGTGCGCCGGTGTACACAGGAAGTGACAATTTTCGCGCGGTTTTAGGCGGATGTTGTAGTAAATTTGGGCGTAACCGAGTAAGATTTGGCCATTTTCGCGGGAAAACTGAATAAGAGGAAGTGAAATCTGAATAATTTTGTGTTACTCATAGCGCGTAATATTTGTCTAGGGCCGCGGGGACTTTGACCGTTTACGTGGAGACTCGCCCAGGTGTTTTTCTCAGGTGTTTTCCGCGTTCCGGGTCAAAGTTGGCGTTTTATTATTATAGTCAGTCGAAGCTTGGATCCGGTACCTCTAGAATTCTCGAGCGGCCGCTAGCGACATCGATCACAAGTTTGTACAAAAAAGCTGAACGAGAAACGTAAAATGATATAAATATCAATATATTAAATTAGATTTTGCATAAAAAACAGACTACATAATACTGTAAAACACAACATATCCAGTCACTATGGCGGCCGCATTAGGCACCCCAGGCTTTACACTTTATGCTTCCGGCTCGTATAATGTGTGGATTTTGAGTTAGGATCCGGCGAGATTTTCAGGAGCTAAGGAAGCTAAAATGGAGAAAAAAATCACTGGATATACCACCGTTGATATATCCCAATGGCATCGTAAAGAACATTTTGAGGCATTTCAGTCAGTTGCTCAATGTACCTATAACCAGACCGTTCAGCTGGATATTACGGCCTTTTTAAAGACCGTAAAGAAAAATAAGCACAAGTTTTATCCGGCCTTTATTCACATTCTTGCCCGCCTGATGAATGCTCATCCGGAATTCCGTATGGCAATGAAAGACGGTGAGCTGGTGATATGGGATAGTGTTCACCCTTGTTACACCGTTTTCCATGAGCAAACTGAAACGTTTTCATCGCTCTGGAGTGAATACCACGACGATTTCCGGCAGTTTCTACACATATATTCGCAAGATGTGGCGTGTTACGGTGAAAACCTGGCCTATTTCCCTAAAGGGTTTATTGAGAATATGTTTTTCGTCTCAGCCAATCCCTGGGTGAGTTTCACCAGTTTTGATTTAAACGTGGCCAATATGGACAACTTCTTCGCCCCCGTTTTCACCATGGGCAAATATTATACGCAAGGCGACAAGGTGCTGATGCCGCTGGCGATTCAGGTTCATCATGCCGTCTGTGATGGCTTCCATGTCGGCAGAATGCTTAATGAATTACAACAGTACTGCGATGAGTGGCAGGGCGGGGCGTAAACGCGTGGATCCGGCTTACTAAAAGCCAGATAACAGTATGCGTATTTGCGCGCTGATTTTTGCGGTATAAGAATATATACTGATATGTATACCCGAAGTATGTCAAAAAGAGGTGTGCTATGAAGCAGCGTATTACAGTGACAGTTGACAGCGACAGCTATCAGTTGCTCAAGGCATATATGATGTCAATATCTCCGGTCTGGTAAGCACAACCATGCAGAATGAAGCCCGTCGTCTGCGTGCCGAACGCTGGAAAGCGGAAAATCAGGAAGGGATGGCTGAGGTCGCCCGGTTTATTGAAATGAACGGCTCTTTTGCTGACGAGAACAGGGACTGGTGAAATGCAGTTTAAGGTTTACACCTATAAAAGAGAGAGCCGTTATCGTCTGTTTGTGGATGTACAGAGTGATATTATTGACACGCCCGGGCGACGGATGGTGATCCCCCTGGCCAGTGCACGTCTGCTGTCAGATAAAGTCTCCCGTGAACTTTACCCGGTGGTGCATATCGGGGATGAAAGCTGGCGCATGATGACCACCGATATGGCCAGTGTGCCGGTCTCCGTTATCGGGGAAGAAGTGGCTGATCTCAGCCACCGCGAAAATGACATCAAAAACGCCATTAACCTGATGTTCTGGGGAATATAAATGTCAGGCTCCGTTATACACAGCCAGTCTGCAGGTCGACCATAGTGACTGGATATGTTGTGTTTTACAGTATTATGTAGTCTGTTTTTTATGCAAAATCTAATTTAATATATTGATATTTATATCATTTTACGTTTCTCGTTCAGCTTTCTTGTACAAAGTGGTGATCGATTCGACAGATCACTGAAATGTGTGGGCGTGGCTTAAGGGTGGGAAAGAATATATAAGGTGGGGGTCTTATGTAGTTTTGTATCTGTTTTGCAGCAGCCGCCGCCGCCATGAGCACCAACTCGTTTGATGGAAGCATTGTGAGCTCATATTTGACAACGCGCATGCCCCCATGGGCCGGGGTGCGTCAGAATGTGATGGGCTCCAGCATTGATGGTCGCCCCGTCCTGCCCGCAAACTCTACTACCTTGACCTACGAGACCGTGTCTGGAACGCCGTTGGAGACTGCAGCCTCCGCCGCCGCTTCAGCCGCTGCAGCCACCGCCCGCGGGATTGTGACTGACTTTGCTTTCCTGAGCCCGCTTGCAAGCAGTGCAGCTTCCCGTTCATCCGCCCGCGATGACAAGTTGACGGCTCTTTTGGCACAATTGGATTCTTTGACCCGGGAACTTAATGTCGTTTCTCAGCAGCTGTTGGATCTGCGCCAGCAGGTTTCTGCCCTGAAGGCTTCCTCCCCTCCCAATGCGGTTTAAAACATAAATAAAAAACCAGACTCTGTTTGGATTTGGATCAAGCAAGTGTCTTGCTGTCTTTATTTAGGGGTTTTGCGCGCGCGGTAGGCCCGGGACCAGCGGTCTCGGTCGTTGAGGGTCCTGTGTATTTTTTCCAGGACGTGGTAAAGGTGACTCTGGATGTTCAGATACATGGGCATAAGCCCGTCTCTGGGGTGGAGGTAGCACCACTGCAGAGCTTCATGCTGCGGGGTGGTGTTGTAGATGATCCAGTCGTAGCAGGAGCGCTGGGCGTGGTGCCTAAAAATGTCTTTCAGTAGCAAGCTGATTGCCAGGGGCAGGCCCTTGGTGTAAGTGTTTACAAAGCGGTTAAGCTGGGATGGGTGCATACGTGGGGATATGAGATGCATCTTGGACTGTATTTTTAGGTTGGCTATGTTCCCAGCCATATCCCTCCGGGGATTCATGTTGTGCAGAACCACCAGCACAGTGTATCCGGTGCACTTGGGAAATTTGTCATGTAGCTTAGAAGGAAATGCGTGGAAGAACTTGGAGACGCCCTTGTGACCTCCAAGATTTTCCATGCATTCGTCCATAATGATGGCAATGGGCCCACGGGCGGCGGCCTGGGCGAAGATATTTCTGGGATCACTAACGTCATAGTTGTGTTCCAGGATGAGATCGTCATAGGCCATTTTTACAAAGCGCGGGCGGAGGGTGCCAGACTGCGGTATAATGGTTCCATCCGGCCCAGGGGCGTAGTTACCCTCACAGATTTGCATTTCCCACGCTTTGAGTTCAGATGGGGGGATCATGTCTACCTGCGGGGCGATGAAGAAAACGGTTTCCGGGGTAGGGGAGATCAGCTGGGAAGAAAGCAGGTTCCTGAGCAGCTGCGACTTACCGCAGCCGGTGGGCCCGTAAATCACACCTATTACCGGGTGCAACTGGTAGTTAAGAGAGCTGCAGCTGCCGTCATCCCTGAGCAGGGGGGCCACTTCGTTAAGCATGTCCCTGACTCGCATGTTTTCCCTGACCAAATCCGCCAGAAGGCGCTCGCCGCCCAGCGATAGCAGTTCTTGCAAGGAAGCAAAGTTTTTCAACGGTTTGAGACCGTCCGCCGTAGGCATGCTTTTGAGCGTTTGACCAAGCAGTTCCAGGCGGTCCCACAGCTCGGTCACCTGCTCTACGGCATCTCGATCCAGCATATCTCCTCGTTTCGCGGGTTGGGGCGGCTTTCGCTGTACGGCAGTAGTCGGTGCTCGTCCAGACGGGCCAGGGTCATGTCTTTCCACGGGCGCAGGGTCCTCGTCAGCGTAGTCTGGGTCACGGTGAAGGGGTGCGCTCCGGGCTGCGCGCTGGCCAGGGTGCGCTTGAGGCTGGTCCTGCTGGTGCTGAAGCGCTGCCGGTCTTCGCCCTGCGCGTCGGCCAGGTAGCATTTGACCATGGTGTCATAGTCCAGCCCCTCCGCGGCGTGGCCCTTGGCGCGCAGCTTGCCCTTGGAGGAGGCGCCGCACGAGGGGCAGTGCAGACTTTTGAGGGCGTAGAGCTTGGGCGCGAGAAATACCGATTCCGGGGAGTAGGCATCCGCGCCGCAGGCCCCGCAGACGGTCTCGCATTCCACGAGCCAGGTGAGCTCTGGCCGTTCGGGGTCAAAAACCAGGTTTCCCCCATGCTTTTTGATGCGTTTCTTACCTCTGGTTTCCATGAGCCGGTGTCCACGCTCGGTGACGAAAAGGCTGTCCGTGTCCCCGTATACAGACTTGAGAGGCCTGTCCTCGAGCGGTGTTCCGCGGTCCTCCTCGTATAGAAACTCGGACCACTCTGAGACAAAGGCTCGCGTCCAGGCCAGCACGAAGGAGGCTAAGTGGGAGGGGTAGCGGTCGTTGTCCACTAGGGGGTCCACTCGCTCCAGGGTGTGAAGACACATGTCGCCCTCTTCGGCATCAAGGAAGGTGATTGGTTTGTAGGTGTAGGCCACGTGACCGGGTGTTCCTGAAGGGGGGCTATAAAAGGGGGTGGGGGCGCGTTCGTCCTCACTCTCTTCCGCATCGCTGTCTGCGAGGGCCAGCTGTTGGGGTGAGTACTCCCTCTGAAAAGCGGGCATGACTTCTGCGCTAAGATTGTCAGTTTCCAAAAACGAGGAGGATTTGATATTCACCTGGCCCGCGGTGATGCCTTTGAGGGTGGCCGCATCCATCTGGTCAGAAAAGACAATCTTTTTGTTGTCAAGCTTGGTGGCAAACGACCCGTAGAGGGCGTTGGACAGCAACTTGGCGATGGAGCGCAGGGTTTGGTTTTTGTCGCGATCGGCGCGCTCCTTGGCCGCGATGTTTAGCTGCACGTATTCGCGCGCAACGCACCGCCATTCGGGAAAGACGGTGGTGCGCTCGTCGGGCACCAGGTGCACGCGCCAACCGCGGTTGTGCAGGGTGACAAGGTCAACGCTGGTGGCTACCTCTCCGCGTAGGCGCTCGTTGGTCCAGCAGAGGCGGCCGCCCTTGCGCGAGCAGAATGGCGGTAGGGGGTCTAGCTGCGTCTCGTCCGGGGGGTCTGCGTCCACGGTAAAGACCCCGGGCAGCAGGCGCGCGTCGAAGTAGTCTATCTTGCATCCTTGCAAGTCTAGCGCCTGCTGCCATGCGCGGGCGGCAAGCGCGCGCTCGTATGGGTTGAGTGGGGGACCCCATGGCATGGGGTGGGTGAGCGCGGAGGCGTACATGCCGCAAATGTCGTAAACGTAGAGGGGCTCTCTGAGTATTCCAAGATATGTAGGGTAGCATCTTCCACCGCGGATGCTGGCGCGCACGTAATCGTATAGTTCGTGCGAGGGAGCGAGGAGGTCGGGACCGAGGTTGCTACGGGCGGGCTGCTCTGCTCGGAAGACTATCTGCCTGAAGATGGCATGTGAGTTGGATGATATGGTTGGACGCTGGAAGACGTTGAAGCTGGCGTCTGTGAGACCTACCGCGTCACGCACGAAGGAGGCGTAGGAGTCGCGCAGCTTGTTGACCAGCTCGGCGGTGACCTGCACGTCTAGGGCGCAGTAGTCCAGGGTTTCCTTGATGATGTCATACTTATCCTGTCCCTTTTTTTTCCACAGCTCGCGGTTGAGGACAAACTCTTCGCGGTCTTTCCAGTACTCTTGGATCGGAAACCCGTCGGCCTCCGAACGGTAAGAGCCTAGCATGTAGAACTGGTTGACGGCCTGGTAGGCGCAGCATCCCTTTTCTACGGGTAGCGCGTATGCCTGCGCGGCCTTCCGGAGCGAGGTGTGGGTGAGCGCAAAGGTGTCCCTGACCATGACTTTGAGGTACTGGTATTTGAAGTCAGTGTCGTCGCATCCGCCCTGCTCCCAGAGCAAAAAGTCCGTGCGCTTTTTGGAACGCGGATTTGGCAGGGCGAAGGTGACATCGTTGAAGAGTATCTTTCCCGCGCGAGGCATAAAGTTGCGTGTGATGCGGAAGGGTCCCGGCACCTCGGAACGGTTGTTAATTACCTGGGCGGCGAGCACGATCTCGTCAAAGCCGTTGATGTTGTGGCCCACAATGTAAAGTTCCAAGAAGCGCGGGATGCCCTTGATGGAAGGCAATTTTTTAAGTTCCTCGTAGGTGAGCTCTTCAGGGGAGCTGAGCCCGTGCTCTGAAAGGGCCCAGTCTGCAAGATGAGGGTTGGAAGCGACGAATGAGCTCCACAGGTCACGGGCCATTAGCATTTGCAGGTGGTCGCGAAAGGTCCTAAACTGGCGACCTATGGCCATTTTTTCTGGGGTGATGCAGTAGAAGGTAAGCGGGTCTTGTTCCCAGCGGTCCCATCCAAGGTTCGCGGCTAGGTCTCGCGCGGCAGTCACTAGAGGCTCATCTCCGCCGAACTTCATGACCAGCATGAAGGGCACGAGCTGCTTCCCAAAGGCCCCCATCCAAGTATAGGTCTCTACATCGTAGGTGACAAAGAGACGCTCGGTGCGAGGATGCGAGCCGATCGGGAAGAACTGGATCTCCCGCCACCAATTGGAGGAGTGGCTATTGATGTGGTGAAAGTAGAAGTCCCTGCGACGGGCCGAACACTCGTGCTGGCTTTTGTAAAAACGTGCGCAGTACTGGCAGCGGTGCACGGGCTGTACATCCTGCACGAGGTTGACCTGACGACCGCGCACAAGGAAGCAGAGTGGGAATTTGAGCCCCTCGCCTGGCGGGTTTGGCTGGTGGTCTTCTACTTCGGCTGCTTGTCCTTGACCGTCTGGCTGCTCGAGGGGAGTTACGGTGGATCGGACCACCACGCCGCGCGAGCCCAAAGTCCAGATGTCCGCGCGCGGCGGTCGGAGCTTGATGACAACATCGCGCAGATGGGAGCTGTCCATGGTCTGGAGCTCCCGCGGCGTCAGGTCAGGCGGGAGCTCCTGCAGGTTTACCTCGCATAGACGGGTCAGGGCGCGGGCTAGATCCAGGTGATACCTAATTTCCAGGGGCTGGTTGGTGGCGGCGTCGATGGCTTGCAAGAGGCCGCATCCCCGCGGCGCGACTACGGTACCGCGCGGCGGGCGGTGGGCCGCGGGGGTGTCCTTGGATGATGCATCTAAAAGCGGTGACGCGGGCGAGCCCCCGGAGGTAGGGGGGGCTCCGGACCCGCCGGGAGAGGGGGCAGGGGCACGTCGGCGCCGCGCGCGGGCAGGAGCTGGTGCTGCGCGCGTAGGTTGCTGGCGAACGCGACGACGCGGCGGTTGATCTCCTGAATCTGGCGCCTCTGCGTGAAGACGACGGGCCCGGTGAGCTTGAGCCTGAAAGAGAGTTCGACAGAATCAATTTCGGTGTCGTTGACGGCGGCCTGGCGCAAAATCTCCTGCACGTCTCCTGAGTTGTCTTGATAGGCGATCTCGGCCATGAACTGCTCGATCTCTTCCTCCTGGAGATCTCCGCGTCCGGCTCGCTCCACGGTGGCGGCGAGGTCGTTGGAAATGCGGGCCATGAGCTGCGAGAAGGCGTTGAGGCCTCCCTCGTTCCAGACGCGGCTGTAGACCACGCCCCCTTCGGCATCGCGGGCGCGCATGACCACCTGCGCGAGATTGAGCTCCACGTGCCGGGCGAAGACGGCGTAGTTTCGCAGGCGCTGAAAGAGGTAGTTGAGGGTGGTGGCGGTGTGTTCTGCCACGAAGAAGTACATAACCCAGCGTCGCAACGTGGATTCGTTGATATCCCCCAAGGCCTCAAGGCGCTCCATGGCCTCGTAGAAGTCCACGGCGAAGTTGAAAAACTGGGAGTTGCGCGCCGACACGGTTAACTCCTCCTCCAGAAGACGGATGAGCTCGGCGACAGTGTCGCGCACCTCGCGCTCAAAGGCTACAGGGGCCTCTTCTTCTTCTTCAATCTCCTCTTCCATAAGGGCCTCCCCTTCTTCTTCTTCTGGCGGCGGTGGGGGAGGGGGGACACGGCGGCGACGACGGCGCACCGGGAGGCGGTCGACAAAGCGCTCGATCATCTCCCCGCGGCGACGGCGCATGGTCTCGGTGACGGCGCGGCCGTTCTCGCGGGGGCGCAGTTGGAAGACGCCGCCCGTCATGTCCCGGTTATGGGTTGGCGGGGGGCTGCCATGCGGCAGGGATACGGCGCTAACGATGCATCTCAACAATTGTTGTGTAGGTACTCCGCCGCCGAGGGACCTGAGCGAGTCCGCATCGACCGGATCGGAAAACCTCTCGAGAAAGGCGTCTAACCAGTCACAGTCGCAAGGTAGGCTGAGCACCGTGGCGGGCGGCAGCGGGCGGCGGTCGGGGTTGTTTCTGGCGGAGGTGCTGCTGATGATGTAATTAAAGTAGGCGGTCTTGAGACGGCGGATGGTCGACAGAAGCACCATGTCCTTGGGTCCGGCCTGCTGAATGCGCAGGCGGTCGGCCATGCCCCAGGCTTCGTTTTGACATCGGCGCAGGTCTTTGTAGTAGTCTTGCATGAGCCTTTCTACCGGCACTTCTTCTTCTCCTTCCTCTTGTCCTGCATCTCTTGCATCTATCGCTGCGGCGGCGGCGGAGTTTGGCCGTAGGTGGCGCCCTCTTCCTCCCATGCGTGTGACCCCGAAGCCCCTCATCGGCTGAAGCAGGGCTAGGTCGGCGACAACGCGCTCGGCTAATATGGCCTGCTGCACCTGCGTGAGGGTAGACTGGAAGTCATCCATGTCCACAAAGCGGTGGTATGCGCCCGTGTTGATGGTGTAAGTGCAGTTGGCCATAACGGACCAGTTAACGGTCTGGTGACCCGGCTGCGAGAGCTCGGTGTACCTGAGACGCGAGTAAGCCCTCGAGTCAAATACGTAGTCGTTGCAAGTCCGCACCAGGTACTGGTATCCCACCAAAAAGTGCGGCGGCGGCTGGCGGTAGAGGGGCCAGCGTAGGGTGGCCGGGGCTCCGGGGGCGAGATCTTCCAACATAAGGCGATGATATCCGTAGATGTACCTGGACATCCAGGTGATGCCGGCGGCGGTGGTGGAGGCGCGCGGAAAGTCGCGGACGCGGTTCCAGATGTTGCGCAGCGGCAAAAAGTGCTCCATGGTCGGGACGCTCTGGCCGGTCAGGCGCGCGCAATCGTTGACGCTCTAGACCGTGCAAAAGGAGAGCCTGTAAGCGGGCACTCTTCCGTGGTCTGGTGGATAAATTCGCAAGGGTATCATGGCGGACGACCGGGGTTCGAGCCCCGTATCCGGCCGTCCGCCGTGATCCATGCGGTTACCGCCCGCGTGTCGAACCCAGGTGTGCGACGTCAGACAACGGGGGAGTGCTCCTTTTGGCTTCCTTCCAGGCGCGGCGGCTGCTGCGCTAGCTTTTTTGGCCACTGGCCGCGCGCAGCGTAAGCGGTTAGGCTGGAAAGCGAAAGCATTAAGTGGCTCGCTCCCTGTAGCCGGAGGGTTATTTTCCAAGGGTTGAGTCGCGGGACCCCCGGTTCGAGTCTCGGACCGGCCGGACTGCGGCGAACGGGGGTTTGCCTCCCCGTCATGCAAGACCCCGCTTGCAAATTCCTCCGGAAACAGGGACGAGCCCCTTTTTTGCTTTTCCCAGATGCATCCGGTGCTGCGGCAGATGCGCCCCCCTCCTCAGCAGCGGCAAGAGCAAGAGCAGCGGCAGACATGCAGGGCACCCTCCCCTCCTCCTACCGCGTCAGGAGGGGCGACATCCGCGGTTGACGCGGCAGCAGATGGTGATTACGAACCCCCGCGGCGCCGGGCCCGGCACTACCTGGACTTGGAGGAGGGCGAGGGCCTGGCGCGGCTAGGAGCGCCCTCTCCTGAGCGGTACCCAAGGGTGCAGCTGAAGCGTGATACGCGTGAGGCGTACGTGCCGCGGCAGAACCTGTTTCGCGACCGCGAGGGAGAGGAGCCCGAGGAGATGCGGGATCGAAAGTTCCACGCAGGGCGCGAGCTGCGGCATGGCCTGAATCGCGAGCGGTTGCTGCGCGAGGAGGACTTTGAGCCCGACGCGCGAACCGGGATTAGTCCCGCGCGCGCACACGTGGCGGCCGCCGACCTGGTAACCGCATACGAGCAGACGGTGAACCAGGAGATTAACTTTCAAAAAAGCTTTAACAACCACGTGCGTACGCTTGTGGCGCGCGAGGAGGTGGCTATAGGACTGATGCATCTGTGGGACTTTGTAAGCGCGCTGGAGCAAAACCCAAATAGCAAGCCGCTCATGGCGCAGCTGTTCCTTATAGTGCAGCACAGCAGGGACAACGAGGCATTCAGGGATGCGCTGCTAAACATAGTAGAGCCCGAGGGCCGCTGGCTGCTCGATTTGATAAACATCCTGCAGAGCATAGTGGTGCAGGAGCGCAGCTTGAGCCTGGCTGACAAGGTGGCCGCCATCAACTATTCCATGCTTAGCCTGGGCAAGTTTTACGCCCGCAAGATATACCATACCCCTTACGTTCCCATAGACAAGGAGGTAAAGATCGAGGGGTTCTACATGCGCATGGCGCTGAAGGTGCTTACCTTGAGCGACGACCTGGGCGTTTATCGCAACGAGCGCATCCACAAGGCCGTGAGCGTGAGCCGGCGGCGCGAGCTCAGCGACCGCGAGCTGATGCACAGCCTGCAAAGGGCCCTGGCTGGCACGGGCAGCGGCGATAGAGAGGCCGAGTCCTACTTTGACGCGGGCGCTGACCTGCGCTGGGCCCCAAGCCGACGCGCCCTGGAGGCAGCTGGGGCCGGACCTGGGCTGGCGGTGGCACCCGCGCGCGCTGGCAACGTCGGCGGCGTGGAGGAATATGACGAGGACGATGAGTACGAGCCAGAGGACGGCGAGTACTAAGCGGTGATGTTTCTGATCAGATGATGCAAGACGCAACGGACCCGGCGGTGCGGGCGGCGCTGCAGAGCCAGCCGTCCGGCCTTAACTCCACGGACGACTGGCGCCAGGTCATGGACCGCATCATGTCGCTGACTGCGCGCAATCCTGACGCGTTCCGGCAGCAGCCGCAGGCCAACCGGCTCTCCGCAATTCTGGAAGCGGTGGTCCCGGCGCGCGCAAACCCCACGCACGAGAAGGTGCTGGCGATCGTAAACGCGCTGGCCGAAAACAGGGCCATCCGGCCCGACGAGGCCGGCCTGGTCTACGACGCGCTGCTTCAGCGCGTGGCTCGTTACAACAGCGGCAACGTGCAGACCAACCTGGACCGGCTGGTGGGGGATGTGCGCGAGGCCGTGGCGCAGCGTGAGCGCGCGCAGCAGCAGGGCAACCTGGGCTCCATGGTTGCACTAAACGCCTTCCTGAGTACACAGCCCGCCAACGTGCCGCGGGGACAGGAGGACTACACCAACTTTGTGAGCGCACTGCGGCTAATGGTGACTGAGACACCGCAAAGTGAGGTGTACCAGTCTGGGCCAGACTATTTTTTCCAGACCAGTAGACAAGGCCTGCAGACCGTAAACCTGAGCCAGGCTTTCAAAAACTTGCAGGGGCTGTGGGGGGTGCGGGCTCCCACAGGCGACCGCGCGACCGTGTCTAGCTTGCTGACGCCCAACTCGCGCCTGTTGCTGCTGCTAATAGCGCCCTTCACGGACAGTGGCAGCGTGTCCCGGGACACATACCTAGGTCACTTGCTGACACTGTACCGCGAGGCCATAGGTCAGGCGCATGTGGACGAGCATACTTTCCAGGAGATTACAAGTGTCAGCCGCGCGCTGGGGCAGGAGGACACGGGCAGCCTGGAGGCAACCCTAAACTACCTGCTGACCAACCGGCGGCAGAAGATCCCCTCGTTGCACAGTTTAAACAGCGAGGAGGAGCGCATTTTGCGCTACGTGCAGCAGAGCGTGAGCCTTAACCTGATGCGCGACGGGGTAACGCCCAGCGTGGCGCTGGACATGACCGCGCGCAACATGGAACCGGGCATGTATGCCTCAAACCGGCCGTTTATCAACCGCCTAATGGACTACTTGCATCGCGCGGCCGCCGTGAACCCCGAGTATTTCACCAATGCCATCTTGAACCCGCACTGGCTACCGCCCCCTGGTTTCTACACCGGGGGATTCGAGGTGCCCGAGGGTAACGATGGATTCCTCTGGGACGACATAGACGACAGCGTGTTTTCCCCGCAACCGCAGACCCTGCTAGAGTTGCAACAGCGCGAGCAGGCAGAGGCGGCGCTGCGAAAGGAAAGCTTCCGCAGGCCAAGCAGCTTGTCCGATCTAGGCGCTGCGGCCCCGCGGTCAGATGCTAGTAGCCCATTTCCAAGCTTGATAGGGTCTCTTACCAGCACTCGCACCACCCGCCCGCGCCTGCTGGGCGAGGAGGAGTACCTAAACAACTCGCTGCTGCAGCCGCAGCGCGAAAAAAACCTGCCTCCGGCATTTCCCAACAACGGGATAGAGAGCCTAGTGGACAAGATGAGTAGATGGAAGACGTACGCGCAGGAGCACAGGGACGTGCCAGGCCCGCGCCCGCCCACCCGTCGTCAAAGGCACGACCGTCAGCGGGGTCTGGTGTGGGAGGACGATGACTCGGCAGACGACAGCAGCGTCCTGGATTTGGGAGGGAGTGGCAACCCGTTTGCGCACCTTCGCCCCAGGCTGGGGAGAATGTTTTAAAAAAAAAAAAGCATGATGCAAAATAAAAAACTCACCAAGGCCATGGCACCGAGCGTTGGTTTTCTTGTATTCCCCTTAGTATGCGGCGCGCGGCGATGTATGAGGAAGGTCCTCCTCCCTCCTACGAGAGTGTGGTGAGCGCGGCGCCAGTGGCGGCGGCGCTGGGTTCTCCCTTCGATGCTCCCCTGGACCCGCCGTTTGTGCCTCCGCGGTACCTGCGGCCTACCGGGGGGAGAAACAGCATCCGTTACTCTGAGTTGGCACCCCTATTCGACACCACCCGTGTGTACCTGGTGGACAACAAGTCAACGGATGTGGCATCCCTGAACTACCAGAACGACCACAGCAACTTTCTGACCACGGTCATTCAAAACAATGACTACAGCCCGGGGGAGGCAAGCACACAGACCATCAATCTTGACGACCGGTCGCACTGGGGCGGCGACCTGAAAACCATCCTGCATACCAACATGCCAAATGTGAACGAGTTCATGTTTACCAATAAGTTTAAGGCGCGGGTGATGGTGTCGCGCTTGCCTACTAAGGACAATCAGGTGGAGCTGAAATACGAGTGGGTGGAGTTCACGCTGCCCGAGGGCAACTACTCCGAGACCATGACCATAGACCTTATGAACAACGCGATCGTGGAGCACTACTTGAAAGTGGGCAGACAGAACGGGGTTCTGGAAAGCGACATCGGGGTAAAGTTTGACACCCGCAACTTCAGACTGGGGTTTGACCCCGTCACTGGTCTTGTCATGCCTGGGGTATATACAAACGAAGCCTTCCATCCAGACATCATTTTGCTGCCAGGATGCGGGGTGGACTTCACCCACAGCCGCCTGAGCAACTTGTTGGGCATCCGCAAGCGGCAACCCTTCCAGGAGGGCTTTAGGATCACCTACGATGATCTGGAGGGTGGTAACATTCCCGCACTGTTGGATGTGGACGCCTACCAGGCGAGCTTGAAAGATGACACCGAACAGGGCGGGGGTGGCGCAGGCGGCAGCAACAGCAGTGGCAGCGGCGCGGAAGAGAACTCCAACGCGGCAGCCGCGGCAATGCAGCCGGTGGAGGACATGAACGATCATGCCATTCGCGGCGACACCTTTGCCACACGGGCTGAGGAGAAGCGCGCTGAGGCCGAAGCAGCGGCCGAAGCTGCCGCCCCCGCTGCGCAACCCGAGGTCGAGAAGCCTCAGAAGAAACCGGTGATCAAACCCCTGACAGAGGACAGCAAGAAACGCAGTTACAACCTAATAAGCAATGACAGCACCTTCACCCAGTACCGCAGCTGGTACCTTGCATACAACTACGGCGACCCTCAGACCGGAATCCGCTCATGGACCCTGCTTTGCACTCCTGACGTAACCTGCGGCTCGGAGCAGGTCTACTGGTCGTTGCCAGACATGATGCAAGACCCCGTGACCTTCCGCTCCACGCGCCAGATCAGCAACTTTCCGGTGGTGGGCGCCGAGCTGTTGCCCGTGCACTCCAAGAGCTTCTACAACGACCAGGCCGTCTACTCCCAACTCATCCGCCAGTTTACCTCTCTGACCCACGTGTTCAATCGCTTTCCCGAGAACCAGATTTTGGCGCGCCCGCCAGCCCCCACCATCACCACCGTCAGTGAAAACGTTCCTGCTCTCACAGATCACGGGACGCTACCGCTGCGCAACAGCATCGGAGGAGTCCAGCGAGTGACCATTACTGACGCCAGACGCCGCACCTGCCCCTACGTTTACAAGGCCCTGGGCATAGTCTCGCCGCGCGTCCTATCGAGCCGCACTTTTTGAGCAAGCATGTCCATCCTTATATCGCCCAGCAATAACACAGGCTGGGGCCTGCGCTTCCCAAGCAAGATGTTTGGCGGGGCCAAGAAGCGCTCCGACCAACACCCAGTGCGCGTGCGCGGGCACTACCGCGCGCCCTGGGGCGCGCACAAACGCGGCCGCACTGGGCGCACCACCGTCGATGACGCCATCGACGCGGTGGTGGAGGAGGCGCGCAACTACACGCCCACGCCGCCACCAGTGTCCACAGTGGACGCGGCCATTCAGACCGTGGTGCGCGGAGCCCGGCGCTATGCTAAAATGAAGAGACGGCGGAGGCGCGTAGCACGTCGCCACCGCCGCCGACCCGGCACTGCCGCCCAACGCGCGGCGGCGGCCCTGCTTAACCGCGCACGTCGCACCGGCCGACGGGCGGCCATGCGGGCCGCTCGAAGGCTGGCCGCGGGTATTGTCACTGTGCCCCCCAGGTCCAGGCGACGAGCGGCCGCCGCAGCAGCCGCGGCCATTAGTGCTATGACTCAGGGTCGCAGGGGCAACGTGTATTGGGTGCGCGACTCGGTTAGCGGCCTGCGCGTGCCCGTGCGCACCCGCCCCCCGCGCAACTAGATTGCAAGAAAAAACTACTTAGACTCGTACTGTTGTATGTATCCAGCGGCGGCGGCGCGCAACGAAGCTATGTCCAAGCGCAAAATCAAAGAAGAGATGCTCCAGGTCATCGCGCCGGAGATCTATGGCCCCCCGAAGAAGGAAGAGCAGGATTACAAGCCCCGAAAGCTAAAGCGGGTCAAAAAGAAAAAGAAAGATGATGATGATGAACTTGACGACGAGGTGGAACTGCTGCACGCTACCGCGCCCAGGCGACGGGTACAGTGGAAAGGTCGACGCGTAAAACGTGTTTTGCGACCCGGCACCACCGTAGTCTTTACGCCCGGTGAGCGCTCCACCCGCACCTACAAGCGCGTGTATGATGAGGTGTACGGCGACGAGGACCTGCTTGAGCAGGCCAACGAGCGCCTCGGGGAGTTTGCCTACGGAAAGCGGCATAAGGACATGCTGGCGTTGCCGCTGGACGAGGGCAACCCAACACCTAGCCTAAAGCCCGTAACACTGCAGCAGGTGCTGCCCGCGCTTGCACCGTCCGAAGAAAAGCGCGGCCTAAAGCGCGAGTCTGGTGACTTGGCACCCACCGTGCAGCTGATGGTACCCAAGCGCCAGCGACTGGAAGATGTCTTGGAAAAAATGACCGTGGAACCTGGGCTGGAGCCCGAGGTCCGCGTGCGGCCAATCAAGCAGGTGGCGCCGGGACTGGGCGTGCAGACCGTGGACGTTCAGATACCCACTACCAGTAGCACCAGTATTGCCACCGCCACAGAGGGCATGGAGACACAAACGTCCCCGGTTGCCTCAGCGGTGGCGGATGCCGCGGTGCAGGCGGTCGCTGCGGCCGCGTCCAAGACCTCTACGGAGGTGCAAACGGACCCGTGGATGTTTCGCGTTTCAGCCCCCCGGCGCCCGCGCGGTTCGAGGAAGTACGGCGCCGCCAGCGCGCTACTGCCCGAATATGCCCTACATCCTTCCATTGCGCCTACCCCCGGCTATCGTGGCTACACCTACCGCCCCAGAAGACGAGCAACTACCCGACGCCGAACCACCACTGGAACCCGCCGCCGCCGTCGCCGTCGCCAGCCCGTGCTGGCCCCGATTTCCGTGCGCAGGGTGGCTCGCGAAGGAGGCAGGACCCTGGTGCTGCCAACAGCGCGCTACCACCCCAGCATCGTTTAAAAGCCGGTCTTTGTGGTTCTTGCAGATATGGCCCTCACCTGCCGCCTCCGTTTCCCGGTGCCGGGATTCCGAGGAAGAATGCACCGTAGGAGGGGCATGGCCGGCCACGGCCTGACGGGCGGCATGCGTCGTGCGCACCACCGGCGGCGGCGCGCGTCGCACCGTCGCATGCGCGGCGGTATCCTGCCCCTCCTTATTCCACTGATCGCCGCGGCGATTGGCGCCGTGCCCGGAATTGCATCCGTGGCCTTGCAGGCGCAGAGACACTGATTAAAAACAAGTTGCATGTGGAAAAATCAAAATAAAAAGTCTGGACTCTCACGCTCGCTTGGTCCTGTAACTATTTTGTAGAATGGAAGACATCAACTTTGCGTCTCTGGCCCCGCGACACGGCTCGCGCCCGTTCATGGGAAACTGGCAAGATATCGGCACCAGCAATATGAGCGGTGGCGCCTTCAGCTGGGGCTCGCTGTGGAGCGGCATTAAAAATTTCGGTTCCACCGTTAAGAACTATGGCAGCAAGGCCTGGAACAGCAGCACAGGCCAGATGCTGAGGGATAAGTTGAAAGAGCAAAATTTCCAACAAAAGGTGGTAGATGGCCTGGCCTCTGGCATTAGCGGGGTGGTGGACCTGGCCAACCAGGCAGTGCAAAATAAGATTAACAGTAAGCTTGATCCCCGCCCTCCCGTAGAGGAGCCTCCACCGGCCGTGGAGACAGTGTCTCCAGAGGGGCGTGGCGAAAAGCGTCCGCGCCCCGACAGGGAAGAAACTCTGGTGACGCAAATAGACGAGCCTCCCTCGTACGAGGAGGCACTAAAGCAAGGCCTGCCCACCACCCGTCCCATCGCGCCCATGGCTACCGGAGTGCTGGGCCAGCACACACCCGTAACGCTGGACCTGCCTCCCCCCGCCGACACCCAGCAGAAACCTGTGCTGCCAGGCCCGACCGCCGTTGTTGTAACCCGTCCTAGCCGCGCGTCCCTGCGCCGCGCCGCCAGCGGTCCGCGATCGTTGCGGCCCGTAGCCAGTGGCAACTGGCAAAGCACACTGAACAGCATCGTGGGTCTGGGGGTGCAATCCCTGAAGCGCCGACGATGCTTCTGAATAGCTAACGTGTCGTATGTGTGTCATGTATGCGTCCATGTCGCCGCCAGAGGAGCTGCTGAGCCGCCGCGCGCCCGCTTTCCAAGATGGCTACCCCTTCGATGATGCCGCAGTGGTCTTACATGCACATCTCGGGCCAGGACGCCTCGGAGTACCTGAGCCCCGGGCTGGTGCAGTTTGCCCGCGCCACCGAGACGTACTTCAGCCTGAATAACAAGTTTAGAAACCCCACGGTGGCGCCTACGCACGACGTGACCACAGACCGGTCCCAGCGTTTGACGCTGCGGTTCATCCCTGTGGACCGTGAGGATACTGCGTACTCGTACAAGGCGCGGTTCACCCTAGCTGTGGGTGATAACCGTGTGCTGGACATGGCTTCCACGTACTTTGACATCCGCGGCGTGCTGGACAGGGGCCCTACTTTTAAGCCCTACTCTGGCACTGCCTACAACGCCCTGGCTCCCAAGGGTGCCCCAAATCCTTGCGAATGGGATGAAGCTGCTACTGCTCTTGAAATAAACCTAGAAGAAGAGGACGATGACAACGAAGACGAAGTAGACGAGCAAGCTGAGCAGCAAAAAACTCACGTATTTGGGCAGGCGCCTTATTCTGGTATAAATATTACAAAGGAGGGTATTCAAATAGGTGTCGAAGGTCAAACACCTAAATATGCCGATAAAACATTTCAACCTGAACCTCAAATAGGAGAATCTCAGTGGTACGAAACTGAAATTAATCATGCAGCTGGGAGAGTCCTTAAAAAGACTACCCCAATGAAACCATGTTACGGTTCATATGCAAAACCCACAAATGAAAATGGAGGGCAAGGCATTCTTGTAAAGCAACAAAATGGAAAGCTAGAAAGTCAAGTGGAAATGCAATTTTTCTCAACTACTGAGGCGACCGCAGGCAATGGTGATAACTTGACTCCTAAAGTGGTATTGTACAGTGAAGATGTAGATATAGAPACCCCAGACACTCATATTTCTTACATGCCCACTATTAAGGAAGGTAACTCACGAGAACTAATGGGCCAACAATCTATGCCCAACAGGCCTAATTACATTGCTTTTAGGGACAATTTTATTGGTCTAATGTATTACAACAGCACGGGTAATATGGGTGTTCTGGCGGGCCAAGCATCGCAGTTGAATGCTGTTGTAGATTTGCAAGACAGAAACACAGAGCTTTCATACCAGCTTTTGCTTGATTCCATTGGTGATAGAACCAGGTACTTTTCTATGTGGAATCAGGCTGTTGACAGCTATGATCCAGATGTTAGAATTATTGAAAATCATGGAACTGAAGATGAACTTCCAAATTACTGCTTTCCACTGGGAGGTGTGATTAATACAGAGACTCTTACCAAGGTAAAACCTAAAACAGGTCAGGAAAATGGATGGGAAAAAGATGCTACAGAATTTTCAGATAAAAATGAAATAAGAGTTGGAAATAATTTTGCCATGGAAATCAATCTAAATGCCAACCTGTGGAGAAATTTCCTGTACTCCAACATAGCGCTGTATTTGCCCGACAAGCTAAAGTACAGTCCTTCCAACGTAAAAATTTCTGATAACCCAAACACCTACGACTACATGAACAAGCGAGTGGTGGCTCCCGGGTTAGTGGACTGCTACATTAACCTTGGAGCACGCTGGTCCCTTGACTATATGGACAACGTCAACCCATTTAACCACCACCGCAATGCTGGCCTGCGCTACCGCTCAATGTTGCTGGGCAATGGTCGCTATGTGCCCTTCCACATCCAGGTGCCTCAGAAGTTCTTTGCCATTAAAAACCTCCTTCTCCTGCCGGGCTCATACACCTACGAGTGGAACTTCAGGAAGGATGTTAACATGGTTCTGCAGAGCTCCCTAGGAAATGACCTAAGGGTTGACGGAGCCAGCATTAAGTTTGATAGCATTTGCCTTTACGCCACCTTCTTCCCCATGGCCCACAACACCGCCTCCACGCTTGAGGCCATGCTTAGAAACGACACCAACGACCAGTCCTTTAACGACTATCTCTCCGCCGCCAACATGCTCTACCCTATACCCGCCAACGCTACCAACGTGCCCATATCCATCCCCTCCCGCAACTGGGCGGCTTTCCGCGGCTGGGCCTTCACGCGCCTTAAGACTAAGGAAACCCCATCACTGGGCTCGGGCTACGACCCTTATTACACCTACTCTGGCTCTATACCCTACCTAGATGGAACCTTTTACCTCAACCACACCTTTAAGAAGGTGGCCATTACCTTTGACTCTTCTGTCAGCTGGCCTGGCAATGACCGCCTGCTTACCCCCAACGAGTTTGAAATTAAGCGCTCAGTTGACGGGGAGGGTTACAACGTTGCCCAGTGTAACATGACCAAAGACTGGTTCCTGGTACAAATGCTAGCTAACTACAACATTGGCTACCAGGGCTTCTATATCCCAGAGAGCTACAAGGACCGCATGTACTCCTTCTTTAGAAACTTCCAGCCCATGAGCCGTCAGGTGGTGGATGATACTAAATACAAGGACTACCAACAGGTGGGCATCCTACACCAACACAACAACTCTGGATTTGTTGGCTACCTTGCCCCCACCATGCGCGAAGGACAGGCCTACCCTGCTAACTTCCCCTATCCGCTTATAGGCAAGACCGCAGTTGACAGCATTACCCAGAAAAAGTTTCTTTGCGATCGCACCCTTTGGCGCATCCCATTCTCCAGTAACTTTATGTCCATGGGCGCACTCACAGACCTGGGCCAAAACCTTCTCTACGCCAACTCCGCCCACGCGCTAGACATGACTTTTGAGGTGGATCCCATGGACGAGCCCACCCTTCTTTATGTTTTGTTTGAAGTCTTTGACGTGGTCCGTGTGCACCGGCCGCACCGCGGCGTCATCGAAACCGTGTACCTGCGCACGCCCTTCTCGGCCGGCAACGCCACAACATAAAGAAGCAAGCAACATCAACAACAGCTGCCGCCATGGGCTCCAGTGAGCAGGAACTGAAAGCCATTGTCAAAGATCTTGGTTGTGGGCCATATTTTTTGGGCACCTATGACAAGCGCTTTCCAGGCTTTGTTTCTCCACACAAGCTCGCCTGCGCCATAGTCAATACGGCCGGTCGCGAGACTGGGGGCGTACACTGGATGGCCTTTGCCTGGAACCCGCACTCAAAAACATGCTACCTCTTTGAGCCCTTTGGCTTTTCTGACCAGCGACTCAAGCAGGTTTACCAGTTTGAGTACGAGTCACTCCTGCGCCGTAGCGCCATTGCTTCTTCCCCCGACCGCTGTATAACGCTGGAAAAGTCCACCCAAAGCGTACAGGGGCCCAACTCGGCCGCCTGTGGACTATTCTGCTGCATGTTTCTCCACGCCTTTGCCAACTGGCCCCAAACTCCCATGGATCACAACCCCACCATGAACCTTATTACCGGGGTACCCAACTCCATGCTCAACAGTCCCCAGGTACAGCCCACCCTGCGTCGCAACCAGGAACAGCTCTACAGCTTCCTGGAGCGCCACTCGCCCTACTTCCGCAGCCACAGTGCGCAGATTAGGAGCGCCACTTCTTTTTGTCACTTGAAAAACATGTAAAAATAATGTACTAGAGACACTTTCAATAAAGGCAAATGCTTTTATTTGTACACTCTCGGGTGATTATTTACCCCCACCCTTGCCGTCTGCGCCGTTTAAAAATCAAAGGGGTTCTGCCGCGCATCGCTATGCGCCACTGGCAGGGACACGTTGCGATACTGGTGTTTAGTGCTCCACTTAAACTCAGGCACAACCATCCGCGGCAGCTCGGTGAAGTTTTCACTCCACAGGCTGCGCACCATCACCAACGCGTTTAGCAGGTCGGGCGCCGATATCTTGAAGTCGCAGTTGGGGCCTCCGCCCTGCGCGCGCGAGTTGCGATACACAGGGTTGCAGCACTGGAACACTATCAGCGCCGGGTGGTGCACGCTGGCCAGCACGCTCTTGTCGGAGATCAGATCCGCGTCCAGGTCCTCCGCGTTGCTCAGGGCGAACGGAGTCAACTTTGGTAGCTGCCTTCCCAAAAAGGGCGCGTGCCCAGGCTTTGAGTTGCACTCGCACCGTAGTGGCATCAAAAGGTGACCGTGCCCGGTCTGGGCGTTAGGATACAGCGCCTGCATAAAAGCCTTGATCTGCTTAAAAGCCACCTGAGCCTTTGCGCCTTCAGAGAAGAACATGCCGCAAGACTTGCCGGAAAACTGATTGGCCGGACAGGCCGCGTCGTGCACGCAGCACCTTGCGTCGGTGTTGGAGATCTGCACCACATTTCGGCCCCACCGGTTCTTCACGATCTTGGCCTTGCTAGACTGCTCCTTCAGCGCGCGCTGCCCGTTTTCGCTCGTCACATCCATTTCAATCACGTGCTCCTTATTTATCATAATGCTTCCGTGTAGACACTTAAGCTCGCCTTCGATCTCAGCGCAGCGGTGCAGCCACAACGCGCAGCCCGTGGGCTCGTGATGCTTGTAGGTCACCTCTGCAAACGACTGCAGGTACGCCTGCAGGAATCGCCCCATCATCGTCACAAAGGTCTTGTTGCTGGTGAAGGTCAGCTGCAACCCGCGGTGCTCCTCGTTCAGCCAGGTCTTGCATACGGCCGCCAGAGCTTCCACTTGGTCAGGCAGTAGTTTGAAGTTCGCCTTTAGATCGTTATCCACGTGGTACTTGTCCATCAGCGCGCGCGCAGCCTCCATGCCCTTCTCCCACGCAGACACGATCGGCACACTCAGCGGGTTCATCACCGTAATTTCACTTTCCGCTTCGCTGGGCTCTTCCTCTTCCTCTTGCGTCCGCATACCACGCGCCACTGGGTCGTCTTCATTCAGCCGCCGCACTGTGCGCTTACCTCCTTTGCCATGCTTGATTAGCACCGGTGGGTTGCTGAAACCCACCATTTGTAGCGCCACATCTTCTCTTTCTTCCTCGCTGTCCACGATTACCTCTGGTGATGGCGGGCGCTCGGGCTTGGGAGAAGGGCGCTTCTTTTTCTTCTTGGGCGCAATGGCCAAATCCGCCGCCGAGGTCGATGGCCGCGGGCTGGGTGTGCGCGGCACCAGCGCGTCTTGTGATGAGTCTTCCTCGTCCTCGGACTCGATACGCCGCCTCATCCGCTTTTTTGGGGGCGCCCGGGGAGGCGGCGGCGACGGGGACGGGGACGACACGTCCTCCATGGTTGGGGGACGTCGCGCCGCACCGCGTCCGCGCTCGGGGGTGGTTTCGCGCTGCTCCTCTTCCCGACTGGCCATTTCCTTCTCCTATAGGCAGAAAAAGATCATGGAGTCAGTCGAGAAGAAGGACAGCCTAACCGCCCCCTCTGAGTTCGCCACCACCGCCTCCACCGATGCCGCCAACGCGCCTACCACCTTCCCCGTCGAGGCACCCCCGCTTGAGGAGGAGGAAGTGATTATCGAGCAGGACCCAGGTTTTGTAAGCGAAGACGACGAGGACCGCTCAGTACCAACAGAGGATAAAAAGCAAGACCAGGACAACGCAGAGGCAAACGAGGAACAAGTCGGGCGGGGGGACGAAAGGCATGGCGACTACCTAGATGTGGGAGACGACGTGCTGTTGAAGCATCTGCAGCGCCAGTGCGCCATTATCTGCGACGCGTTGCAAGAGCGCAGCGATGTGCCCCTCGCCATAGCGGATGTCAGCCTTGCCTACGAACGCCACCTATTCTCACCGCGCGTACCCCCCAAACGCCAAGAAAACGGCACATGCGAGCCCAACCCGCGCCTCAACTTCTACCCCGTATTTGCCGTGCCAGAGGTGCTTGCCACCTATCACATCTTTTTCCAAAACTGCAAGATACCCCTATCCTGCCGTGCCAACCGCAGCCGAGCGGACAAGCAGCTGGCCTTGCGGCAGGGCGCTGTCATACCTGATATCGCCTCGCTCAACGAAGTGCCAAAAATCTTTGAGGGTCTTGGACGCGACGAGAAGCGCGCGGCAAACGCTCTGCAACAGGAAAACAGCGAAAATGAAAGTCACTCTGGAGTGTTGGTGGAACTCGAGGGTGACAACGCGCGCCTAGCCGTACTAAAACGCAGCATCGAGGTCACCCACTTTGCCTACCCGGCACTTAACCTACCCCCCAAGGTCATGAGCACAGTCATGAGTGAGCTGATCGTGCGCCGTGCGCAGCCCCTGGAGAGGGATGCAAATTTGCAAGAACAAACAGAGGAGGGCCTACCCGCAGTTGGCGACGAGCAGCTAGCGCGCTGGCTTCAAACGCGCGAGCCTGCCGACTTGGAGGAGCGACGCAAACTAATGATGGCCGCAGTGCTCGTTACCGTGGAGCTTGAGTGCATGCAGCGGTTCTTTGCTGACCCGGAGATGCAGCGCAAGCTAGAGGAAACATTGCACTACACCTTTCGACAGGGCTACGTACGCCAGGCCTGCAAGATCTCCAACGTGGAGCTCTGCAACCTGGTCTCCTACCTTGGAATTTTGCACGAAAACCGCCTTGGGCAAAACGTGCTTCATTCCACGCTCAAGGGCGAGGCGCGCCGCGACTACGTCCGCGACTGCGTTTACTTATTTCTATGCTACACCTGGCAGACGGCCATGGGCGTTTGGCAGCAGTGCTTGGAGGAGTGCAACCTCAAGGAGCTGCAGAAACTGCTAAAGCAAAACTTGAAGGACCTATGGACGGCCTTCAACGAGCGCTCCGTGGCCGCGCACCTGGCGGACATCATTTTCCCCGAACGCCTGCTTAAAACCCTGCAACAGGGTCTGCCAGACTTCACCAGTCAAAGCATGTTGCAGAACTTTAGGAACTTTATCCTAGAGCGCTCAGGAATCTTGCCCGCCACCTGCTGTGCACTTCCTAGCGACTTTGTGCCCATTAAGTACCGCGAATGCCCTCCGCCGCTTTGGGGCCACTGCTACCTTCTGCAGCTAGCCAACTACCTTGCCTACCACTCTGACATAATGGAAGACGTGAGCGGTGACGGTCTACTGGAGTGTCACTGTCGCTGCAACCTATGCACCCCGCACCGCTCCCTGGTTTGCAATTCGCAGCTGCTTAACGAAAGTCAAATTATCGGTACCTTTGAGCTGCAGGGTCCCTCGCCTGACGAAAAGTCCGCGGCTCCGGGGTTGAAACTCACTCCGGGGCTGTGGACGTCGGCTTACCTTCGCAAATTTGTACCTGAGGACTACCACGCCCACGAGATTAGGTTCTACGAAGACCAATCCCGCCCGCCAAATGCGGAGCTTACCGCCTGCGTCATTACCCAGGGCCACATTCTTGGCCAATTGCAAGCCATCAACAAAGCCCGCCAAGAGTTTCTGCTACGAAAGGGACGGGGGGTTTACTTGGACCCCCAGTCCGGCGAGGAGCTCAACCCAATCCCCCCGCCGCCGCAGCCCTATCAGCAGCAGCCGCGGGCCCTTGCTTCCCAGGATGGCACCCAAAAAGAAGCTGCAGCTGCCGCCGCCACCCACGGACGAGGAGGAATACTGGGACAGTCAGGCAGAGGAGGTTTTGGACGAGGAGGAGGAGGACATGATGGAAGACTGGGAGAGCCTAGACGAGGAAGCTTCCGAGGTCGAAGAGGTGTCAGACGAAACACCGTCACCCTCGGTCGCATTCCCCTCGCCGGCGCCCCAGAAATCGGCAACCGGTTCCAGCATGGCTACAACCTCCGCTCCTCAGGCGCCGCCGGCACTGCCCGTTCGCCGACCCAACCGTAGATGGGACACCACTGGAACCAGGGCCGGTAAGTCCAAGCAGCCGCCGCCGTTAGCCCAAGAGCAACAACAGCGCCAAGGCTACCGCTCATGGCGCGGGCACAAGAACGCCATAGTTGCTTGCTTGCAAGACTGTGGGGGCAACATCTCCTTCGCCCGCCGCTTTCTTCTCTACCATCACGGCGTGGCCTTCCCCCGTAACATCCTGCATTACTACCGTCATCTCTACAGCCCATACTGCACCGGCGGCAGCGGCAGCGGCAGCAACAGCAGCGGCCACACAGAAGCAAAGGCGACCGGATAGCAAGACTCTGACAAAGCCCAAGAAATCCACAGCGGCGGCAGCAGCAGGAGGAGGAGCGCTGCGTCTGGCGCCCAACGAACCCGTATCGACCCGCGAGCTTAGAAACAGGATTTTTCCCACTCTGTATGCTATATTTCAACAGAGCAGGGGCCAAGAACAAGAGCTGAAAATAAAAAACAGGTCTCTGCGATCCCTCACCCGCAGCTGCCTGTATCACAAAAGCGAAGATCAGCTTCGGCGCACGCTGGAAGACGCGGAGGCTCTCTTCAGTAAATACTGCGCGCTGACTCTTAAGGACTAGTTTCGCGCCCTTTCTCAAATTTAAGCGCGAAAACTACGTCATCTCCAGCGGCCACACCCGGCGCCAGCACCTGTCGTCAGCGCCATTATGAGCAAGGAAATTCCCACGCCCTACATGTGGAGTTACCAGCCACAAATGGGACTTGCGGCTGGAGCTGCCCAAGACTACTCAACCCGAATAAACTACATGAGCGCGGGACCCCACATGATATCCCGGGTCAACGGAATCCGCGCCCACCGAAACCGAATTCTCTTGGAACAGGCGGCTATTACCACCACACCTCGTAATAACCTTAATCCCCGTAGTTGGCCCGCTGCCCTGGTGTACCAGGAAAGTCCCGCTCCCACCACTGTGGTACTTCCCAGAGACGCCCAGGCCGAAGTTCAGATGACTAACTCAGGGGCGCAGCTTGCGGGCGGCTTTCGTCACAGGGTGCGGTCGCCCGGGCAGGGTATAACTCACCTGACAATCAGAGGGCGAGGTATTCAGCTCAACGACGAGTCGGTGAGCTCCTCGCTTGGTCTCCGTCCGGACGGGACATTTCAGATCGGCGGCGCCGGCCGTCCTTCATTCACGCCTCGTCAGGCAATCCTAACTCTGCAGACCTCGTCCTCTGAGCCGCGCTCTGGAGGCATTGGAACTCTGCAATTTATTGAGGAGTTTGTGCCATCGGTCTACTTTAACCCCTTCTCGGGACCTCCCGGCCACTATCCGGATCAATTTATTCCTAACTTTGACGCGGTAAAGGACTCGGCGGACGGCTACGACTGAATGTTAAGTGGAGAGGCAGAGCAACTGCGCCTGAAACACCTGGTCCACTGTCGCCGCCACAAGTGCTTTGCCCGCGACTCCGGTGAGTTTTGCTACTTTGAATTGCCCGAGGATCATATCGAGGGCCCGGCGCACGGCGTCCGGCTTACCGCCCAGGGAGAGCTTGCCCGTAGCCTGATTCGGGAGTTTACCCAGCGCCCCCTGCTAGTTGAGCGGGACAGGGGACCCTGTGTTCTCACTGTGATTTGCAACTGTCCTAACCTTGGATTACATCAAGATCTTTGTTGCCATCTCTGTGCTGAGTATAATAAATACAGAAATTAAAATATACTGGGGCTCCTATCGCCATCCTGTAAACGCCACCGTCTTCACCCGCCCAAGCAAACCAAGGCGAACCTTACCTGGTACTTTTAACATCTCTCCCTCTGTGATTTACAACAGTTTCAACCCAGACGGAGTGAGTCTACGAGAGAACCTCTCCGAGCTCAGCTACTCCATCAGAAAAAACACCACCCTCCTTACCTGCCGGGAACGTACGAGTGCGTCACCGGCCGCTGCACCACACCTACCGCCTGACCGTAAACCAGACTTTTTCCGGACAGACCTCAATAACTCTGTTTACCAGAACAGGAGGTGAGCTTAGAAAACCCTTAGGGTATTAGGCCAAAGGCGCAGCTACTGTGGGGTTTATGAACAATTCAAGCAACTCTACGGGCTATTCTAATTCAGGTTTCTCTAGAAATGGACGGAATTATTACAGAGCAGCGCCTGCTAGAAAGACGCAGGGCAGCGGCCGAGCAACAGCGCATGAATCAAGAGCTCCAAGACATGGTTAACTTGCACCAGTGCAAAAGGGGTATCTTTTGTCTGGTAAAGCAGGCCAAAGTCACCTACGACAGTAATACCACCGGACACCGCCTTAGCTACAAGTTGCCAACCAAGCGTCAGAPATTGGTGGTCATGGTGGGAGAAAAGCCCATTACCATAACTCAGCACTCGGTAGAAACCGAAGGCTGCATTCACTCACCTTGTCAAGGACCTGAGGATCTCTGCACCCTTATTAAGACCCTGTGCGGTCTCAAAGATCTTATTCCCTTTAACTAATAAAAAAAAATAATAAAGCATCACTTACTTAAAATCAGTTAGCAAATTTCTGTCCAGTTTATTCAGCAGCACCTCCTTGCCCTCCTCCCAGCTCTGGTATTGCAGCTTCCTCCTGGCTGCAAACTTTCTCCACAATCTAAATGGAATGTCAGTTTCCTCCTGTTCCTGTCCATCCGCACCCACTATCTTCATGTTGTTGCAGATGAAGCGCGCAAGACCGTCTGAAGATACCTTCAACCCCGTGTATCCATATGACACGGAAACCGGTCCTCCAACTGTGCCTTTTCTTACTCCTCCCTTTGTATCCCCCAATGGGTTTCAAGAGAGTCCCCCTGGGGTACTCTCTTTGCGCCTATCCGAACCTCTAGTTACCTCCAATGGCATGCTTGCGCTCAAAATGGGCAACGGCCTCTCTCTGGACGAGGCCGGCAACCTTACCTCCCAAAATGTAACCACTGTGAGCCCACCTCTCAAAAAAACCAAGTCAAACATAAACCTGGAAATATCTGCACCCCTCACAGTTACCTCAGAAGCCCTAACTGTGGCTGCCGCCGCACCTCTAATGGTCGCGGGCAACACACTCACCATGCAATCACAGGCCCCGCTAACCGTGCACGACTCCAAACTTAGCATTGCCACCCAAGGACCCCTCACAGTGTCAGAAGGAAAGCTAGCCCTGCAAACATCAGGCCCCCTCACCACCACCGATAGCAGTACCCTTACTATCACTGCCTCACCCCCTCTAACTACTGCCACTGGTAGCTTGGGCATTGACTTGAAAGAGCCCATTTATACACAAAATGGAAAACTAGGACTAAAGTACGGGGCTCCTTTGCATGTAACAGACGACCTAAACACTTTGACCGTAGCAACTGGTCCAGGTGTGACTATTAATAATACTTCCTTGCAAACTAAAGTTACTGGAGCCTTGGGTTTTGATTCACAAGGCAATATGCAACTTAATGTAGCAGGAGGACTAAGGATTGATTCTCAAAACAGACGCCTTATACTTGATGTTAGTTATCCGTTTGATGCTCAAAACCAACTAAATCTAAGACTAGGACAGGGCCCTCTTTTTATAAACTCAGCCCACAACTTGGATATTAACTACAACAAAGGCCTTTACTTGTTTACAGCTTCAAACAATTCCAAAAAGCTTGAGGTTAACCTAAGCACTGCCAAGGGGTTGATGTTTGACGCTACAGCCATAGCCATTAATGCAGGAGATGGGCTTGAATTTGGTTCACCTAATGCACCAAACACAAATCCCCTCAAAACAAAAATTGGCCATGGCCTAGAATTTGATTCAAACAAGGCTATGGTTCCTAAACTAGGAACTGGCCTTAGTTTTGACAGCACAGGTGCCATTACAGTAGGAAACAAAAATAATGATAAGCTAACTTTGTGGACCACACCAGCTCCATCTCCTAACTGTAGACTAAATGCAGAGAAAGATGCTAAACTCACTTTGGTCTTAACAAAATGTGGCAGTCAAATACTTGCTACAGTTTCAGTTTTGGCTGTTAAAGGCAGTTTGGCTCCAATATCTGGAACAGTTCAAAGTGCTCATCTTATTATAAGATTTGACGAAAATGGAGTGCTACTAAACAATTCCTTCCTGGACCCAGAATATTGGAACTTTAGAAATGGAGATCTTACTGAAGGCACAGCCTATACAAACGCTGTTGGATTTATGCCTAACCTATCAGCTTATCCAAAATCTCACGGTAAAACTGCCAAAAGTAACATTGTCAGTCAAGTTTACTTAAACGGAGACAAAACTAAACCTGTAACACTAACCATTACACTAAACGGTACACAGGAAACAGGAGACACAACTCCAAGTGCATACTCTATGTCATTTTCATGGGACTGGTCTGGCCACAACTACATTAATGAAATATTTGCCACATCCTCTTACACTTTTTCATACATTGCCCAAGAATAAAGAATCGTTTGTGTTATGTTTCAACGTGTTTATTTTTCAATTGCAGAAAATTTCGAATCATTTTTCATTCAGTAGTATAGCCCCACCACCACATAGCTTATACAGATCACCGTACCTTAATCAAACTCACAGAACCCTAGTATTCAACCTGCCACCTCCCTCCCAACACACAGAGTACACAGTCCTTTCTCCCCGGCTGGCCTTAAAAAGCATCATATCATGGGTAACAGACATATTCTTAGGTGTTATATTCCACACGGTTTCCTGTCGAGCCAAACGCTCATCAGTGATATTAATAAACTCCCCGGGCAGCTCACTTAAGTTCATGTCGCTGTCCAGCTGCTGAGCCACAGGCTGCTGTCCAACTTGCGGTTGCTTAACGGGCGGCGAAGGAGAAGTCCACGCCTACATGGGGGTAGAGTCATAATCGTGCATCAGGATAGGGCGGTGGTGCTGCAGCAGCGCGCGAATAAACTGCTGCCGCCGCCGCTCCGTCCTGCAGGAATACAACATGGCAGTGGTCTCCTCAGCGATGATTCGCACCGCCCGCAGCATAAGGCGCCTTGTCCTCCGGGCACAGCAGCGCACCCTGATCTCACTTAAATCAGCACAGTAACTGCAGCACAGCACCACAATATTGTTCAAAATCCCACAGTGCAAGGCGCTGTATCCAAAGCTCATGGCGGGGACCACAGAACCCACGTGGCCATCATACCACAAGCGCAGGTAGATTAAGTGGCGACCCCTCATAAACACGCTGGACATAAACATTACCTCTTTTGGCATGTTGTAATTCACCACCTCCCGGTACCATATAAACCTCTGATTAAACATGGCGCCATCCACCACCATCCTAAACCAGCTGGCCAAAACCTGCCCGCCGGCTATACACTGCAGGGAACCGGGACTGGAACAATGACAGTGGAGAGCCCAGGACTCGTAACCATGGATCATCATGCTCGTCATGATATCAATGTTGGCACAACACAGGCACACGTGCATACACTTCCTCAGGATTACAAGCTCCTCCCGCGTTAGAACCATATCCCAGGGAACAACCCATTCCTGAATCAGCGTAAATCCCACACTGCAGGGAAGACCTCGCACGTAACTCACGTTGTGCATTGTCAAAGTGTTACATTCGGGCAGCAGCGGATGATCCTCCAGTATGGTAGCGCGGGTTTCTGTCTCAAAAGGAGGTAGACGATCCCTACTGTACGGAGTGCGCCGAGACAACCGAGATCGTGTTGGTCGTAGTGTCATGCCAATGGAAACGCCGGACGTAGTCATATTTCCTGAAGCAAAACCAGGTGCGGGCGTGACAAACAGATCTGCGTCTCCGGTCTCGCCGCTTAGATCGCTCTGTGTAGTAGTTGTAGTATATCCACTCTCTCAAAGCATCCAGGCGCCCCCTGGCTTCGGGTTCTATGTAAACTCCTTCATGCGCCGCTGCCCTGATAACATCCACCACCGCAGAATAAGCCACACCCAGCCAACCTACACATTCGTTCTGCGAGTCACACACGGGAGGAGCGGGAAGAGCTGGAAGAACCATGTTTTTTTTTTTATTCCAAAAGATTATCCAAAACCTCAAAATGAAGATCTATTAAGTGAACGCGCTCCCCTCCGGTGGCGTGGTCAAACTCTACAGCCAAAGAACAGATAATGGCATTTGTAAGATGTTGCACAATGGCTTCCAAAAGGCAAACGGCCCTCACGTCCAAGTGGACGTAAAGGCTAAACCCTTCAGGGTGAATCTCCTCTATAAACATTCCAGCACCTTCAACCATGCCCAAATAATTCTCATCTCGCCACCTTCTCAATATATCTCTAAGCAAATCCCGAATATTAAGTCCGGCCATTGTAAAAATCTGCTCCAGAGCGCCCTCCACCTTCAGCCTCAAGCAGCGAATCATGATTGCAAAAATTCAGGTTCCTCACAGACCTGTATAAGATTCAAAAGCGGAACATTAACAAAAATACCGCGATCCCGTAGGTCCCTTCGCAGGGCCAGCTGAACATAATCGTGCAGGTCTGCACGGACCAGCGCGGCCACTTCCCCGCCAGGAACCTTGACAAAAGAACCCACACTGATTATGACACGCATACTCGGAGCTATGCTAACCAGCGTAGCCCCGATGTAAGCTTTGTTGCATGGGCGGCGATATAAAATGCAAGGTGCTGCTCAAAAAATCAGGCAAAGCCTCGCGCAAAAAAGAAAGCACATCGTAGTCATGCTCATGCAGATAAAGGCAGGTAAGCTCCGGAACCACCACAGAAAAAGACACCATTTTTCTCTCAAACATGTCTGCGGGTTTCTGCATAAACACAAAATAAAATAACAAAAAAACATTTAAACATTAGAAGCCTGTCTTACAACAGGAAAAACAACCCTTATAAGCATAAGACGGACTACGGCCATGCCGGCGTGACCGTAAAAAAACTGGTCACCGTGATTAAAAAGCACCACCGACAGCTCCTCGGTCATGTCCGGAGTCATAATGTAAGACTCGGTAAACACATCAGGTTGATTCACATCGGTCAGTGCTAAAAAGCGACCGAAATAGCCCGGGGGAATACATACCCGCAGGCGTAGAGACAACATTACAGCCCCCATAGGAGGTATAACAAAATTAATAGGAGAGAAAAACACATAAACACCTGAAAAACCCTCCTGCCTAGGCAAAATAGCACCCTCCCGCTCCAGAACAACATACAGCGCTTCCACAGCGGCAGCCATAACAGTCAGCCTTACCAGTAAAAAAGAAAACCTATTAAAAAAACACCACTCGACACGGCACCAGCTCAATCAGTCACAGTGTAAAAAAGGGCCAAGTGCAGAGCGAGTATATATAGGACTAAAAAATGACGTAACGGTTAAAGTCCACAAAAAACACCCAGAAAACCGCACGCGAACCTACGCCCAGAAACGAAAGCCAAAAAACCCACAACTTCCTCAAATCGTCACTTCCGTTTTCCCACGTTACGTCACTTCCCATTTTAAGAAAACTACAATTCCCAACACATACAAGTTACTCCGCCCTAAAACCTACGTCACCCGCCCCGTTCCCACGCCCCGCGCCACGTCACAAACTCCACCCCCTCATTATCATATTGGCTTCAATCCAAAATAAGGTATATTATTGATGATGTTAATTAATTTAAATCCGCATGCGATATCGAGCTCTCCCGGGAATTCGGATCTGCGACGCGAGGCTGGATGGCCTTCCCCATTATGATTCTTCTCGCTTCCGGCGGCATCGGGATGCCCGCGTTGCAGGCCATGCTGTCCAGGCAGGTAGATGACGACCATCAGGGACAGCTTCACGGCCAGCAAAAGGCCAGGAACCGTAAAAAGGCCGCGTTGCTGGCGTTTTTCCATAGGCTCCGCCCCCCTGACGAGCATCACAAAAATCGACGCTCAAGTCAGAGGTGGCGAAACCCGACAGGACTATAAAGATACCAGGCGTTTCCCCCTGGAAGCTCCCTCGTGCGCTCTCCTGTTCCGACCCTGCCGCTTACCGGATACCTGTCCGCCTTTCTCCCTTCGGGAAGCGTGGCGCTTTCTCAATGCTCACGCTGTAGGTATCTCAGTTCGGTGTAGGTCGTTCGCTCCAAGCTGGGCTGTGTGCACGAACCCCCCGTTCAGCCCGACCGCTGCGCCTTATCCGGTAACTATCGTCTTGAGTCCAACCCGGTAAGACACGACTTATCGCCACTGGCAGCAGCCACTGGTAACAGGATTAGCAGAGCGAGGTATGTAGGCGGTGCTACAGAGTTCTTGAAGTGGTGGCCTAACTACGGCTACACTAGAAGGACAGTATTTGGTATCTGCGCTCTGCTGAAGCCAGTTACCTTCGGAAAAAGAGTTGGTAGCTCTTGATCCGGCAAACAAACCACCGCTGGTAGCGGTGGTTTTTTTGTTTGCAAGCAGCAGATTACGCGCAGAAAAAAAGGATCTCAAGAAGATCCTTTGATCTTTTCTACGGGGTCTGACGCTCAGTGGAACGAAAACTCACGTTAAGGGATTTTGGTCATGAGATTATCAAAAAGGATCTTCACCTAGATCCTTTTAAATCAATCTAAAGTATATATGAGTAAACTTGGTCTGACAGTTACCAATGCTTAATCAGTGAGGCACCTATCTCAGCGATCTGTCTATTTCGTTCATCCATAGTTGCCTGACTCCCCGTCGTGTAGATAACTACGATACGGGAGGGCTTACCATCTGGCCCCAGTGCTGCAATGATACCGCGAGACCCACGCTCACCGGCTCCAGATTTATCAGCAATAAACCAGCCAGCCGGAAGGGCCGAGCGCAGAAGTGGTCCTGCAACTTTATCCGCCTCCATCCAGTCTATTAATTGTTGCCGGGAAGCTAGAGTAAGTAGTTCGCCAGTTAATAGTTTGCGCAACGTTGTTGCCATTGNTGCAGGCATCGTGGTGTCACGCTCGTCGTTTGGTATGGCTTCATTCAGCTCCGGTTCCCAACGATCAAGGCGAGTTACATGATCCCCCATGTTGTGCAAAAAAGCGGTTAGCTCCTTCGGTCCTCCGATCGTTGTCAGAAGTAAGTTGGCCGCAGTGTTATCACTCATGGTTATGGCAGCACTGCATAATTCTCTTACTGTCATGCCATCCGTAAGATGCTTTTCTGTGACTGGTGAGTACTCAACCAAGTCATTCTGAGAATAGTGTATGCGGCGACCGAGTTGCTCTTGCCCGGCGTCAACACGGGATAATACCGCGCCACATAGCAGAACTTTAAAAGTGCTCATCATTGGAAAACGTTCTTCGGGGCGAAAACTCTCAAGGATCTTACCGCTGTTGAGATCCAGTTCGATGTAACCCACTCGTGCACCCAACTGATCTTCAGCATCTTTTACTTTCACCAGCGTTTCTGGGTGAGCAAAAACAGGAAGGCAAAATGCCGCAAAAAAGGGAATAAGGGCGACACGGAAATGTTGAATACTCATACTCTTCCTTTTTCAATATTATTGAAGCATTTATCAGGGTTATTGTCTCATGAGCGGATACATATTTGAATGTATTTAGAAAAATAAACAAATAGGGGTTCCGCGCACATTTCCCCGAAAAGTGCCACCTGACGTCTAAGAAACCATTATTATCATGACATTAACCTATAAAAATAGGCGTATCACGAGGCCCTTTCGTCTTCAAGGATCCGAATTCCCGGGAGAGCTCGATATCGCATGCGGATTTAAATTAATTAA

[1243] TABLE 11 Nucleotide sequence of pAd/CMV/V5-GW/lacZ.PL-DEST ™.CATCATCAATAATATACCTTATTTTGGATTGAAGCCAATATGATAATGAGGGGGTGGAGTTTGTGACGTGGCGCGGGGCGTGGGAACGGGGCGGGTGACGTAGTAGTGTGGCGGAAGTGTGATGTTGCAAGTGTGGCGGAACACATGTAAGCGACGGATGTGGCAAAAGTGACGTTTTTGGTGTGCGCCGGTGTACACAGGAAGTGACAATTTTCGCGCGGTTTTAGGCGGATGTTGTAGTAAATTTGGGCGTAACCGAGTAAGATTTGGCCATTTTCGCGGGAAAACTGAATAAGAGGAAGTGAAATCTGAATAATTTTGTGTTACTCATAGCGCGTAATATTTGTCTAGGGCCGCGGGGACTTTGACCGTTTACGTGGAGACTCGCCCAGGTGTTTTTCTCAGGTGTTTTCCGCGTTCCGGGTCAAAGTTGGCGTTTTATTATTATAGTCAGTCGAAGCTTGGATCCGGTACCTCTAGAATTCTCGAGCGGCCGCTAGCGACATCGGATCTCCCGATCCCCTATGGTCGACTCTCAGTACAATCTGCTCTGATGCCGCATAGTTAAGCCAGTATCTGCTCCCTGCTTGTGTGTTGGAGGTCGCTGAGTAGTGCGCGAGCAAAATTTAAGCTACAACAAGGCAAGGCTTGACCGACAATTGCATGAAGAATCTGCTTAGGGTTAGGCGTTTTGCGCTGCTTCGCGATGTACGGGCCAGATATACGCGTTGACATTGATTATTGACTAGTTATTAATAGTAATCAATTACGGGGTCATTAGTTCATAGCCCATATATGGAGTTCCGCGTTACATAACTTACGGTAAATGGCCCGCCTGGCTGACCGCCCAACGACCCCCGCCCATTGACGTCAATAATGACGTATGTTCCCATAGTAACGCCAATAGGGACTTTCCATTGACGTCAATGGGTGGACTATTTACGGTAAACTGCCCACTTGGCAGTACATCAAGTGTATCATATGCCAAGTACGCCCCCTATTGACGTCAATGACGGTAAATGGCCCGCCTGGCATTATGCCCAGTACATGACCTTATGGGACTTTCCTACTTGGCAGTACATCTACGTATTAGTCATCGCTATTACCATGGTGATGCGGTTTTGGCAGTACATCAATGGGCGTGGATAGCGGTTTGACTCACGGGGATTTCCAAGTCTCCACCCCATTGACGTCAATGGGAGTTTGTTTTGGCACCAAAATCAACGGGACTTTCCAAAATGTCGTAACAACTCCGCCCCATTGACGCAAATGGGCGGTAGGCGTGTACGGTGGGAGGTCTATATAAGCAGAGCTCTCTGGCTAACTAGAGAACCCACTGCTTACTGGCTTATCGAAATTAATACGACTCACTATAGGGAGACCCAAGCTGGCTAGTTAAGCTATCAACAAGTTTGTACAAAAAAGCAGGCTCCGCGGCCGCCCCCTTCACCATGATAGATCCCGTCGTTTTACAACGTCGTGACTGGGAAAACCCTGGCGTTACCCAACTTAATCGCCTTGCAGCACATCCCCCTTTCGCCAGCTGGCGTAATAGCGAAGAGGCCCGCACCGATCGCCCTTCCCAACAGTTGCGCAGCCTGAATGGCGAATGGCGCTTTGCCTGGTTTCCGGCACCAGAAGCGGTGCCGGAAAGCTGGCTGGAGTGCGATCTTCCTGAGGCCGATACTGTCGTCGTCCCCTCAAACTGGCAGATGCACGGTTACGATGCGCCCATCTACACCAACGTAACCTATCCCATTACGGTCAATCCGCCGTTTGTTCCCACGGAGAATCCGACGGGTTGTTACTCGCTCACATTTAATGTTGATGAAAGCTGGCTACAGGAAGGCCAGACGCGAATTATTTTTGATGGCGTTAACTCGGCGTTTCATCTGTGGTGCAACGGGCGCTGGGTCGGTTACGGCCAGGACAGTCGTTTGCCGTCTGAATTTGACCTGAGCGCATTTTTACGCGCCGGAGAAAACCGCCTCGCGGTGATGGTGCTGCGTTGGAGTGACGGCAGTTATCTGGAAGATCAGGATATGTGGCGGATGAGCGGCATTTTCCGTGACGTCTCGTTGCTGCATAAACCGACTACACAAATCAGCGATTTCCATGTTGCCACTCGCTTTAATGATGATTTCAGCCGCGCTGTACTGGAGGCTGAAGTTCAGATGTGCGGCGAGTTGCGTGACTACCTACGGGTAACAGTTTCTTTATGGCAGGGTGAAACGCAGGTCGCCAGCGGCACCGCGCCTTTCGGCGGTGAAATTATCGATGAGCGTGGTGGTTATGCCGATCGCGTCACACTACGTCTGAACGTCGAAAACCCGAAACTGTGGAGCGCCGAAATCCCGAATCTCTATCGTGCGGTGGTTGAACTGCACACCGCCGACGGCACGCTGATTGAAGCAGAAGCCTGCGATGTCGGTTTCCGCGAGGTGCGGATTGAAAATGGTCTGCTGCTGCTGAACGGCAAGCCGTTGCTGATTCGAGGCGTTAACCGTCACGAGCATCATCCTCTGCATGGTCAGGTCATGGATGAGCAGACGATGGTGCAGGATATCCTGCTGATGAAGCAGAACAACTTTAACGCCGTGCGCTGTTCGCATTATCCGAACCATCCGCTGTGGTACACGCTGTGCGACCGCTACGGCCTGTATGTGGTGGATGAAGCCAATATTGAAACCCACGGCATGGTGCCAATGAATCGTCTGACCGATGATCCGCGCTGGCTACCGGCGATGAGCGAACGCGTAACGCGAATGGTGCAGCGCGATCGTAATCACCCGAGTGTGATCATCTGGTCGCTGGGGAATGAATCAGGCCACGGCGCTAATCACGACGCGCTGTATCGCTGGATCAAATCTGTCGATCCTTCCCGCCCGGTGCAGTATGAAGGCGGCGGAGCCGACACCACGGCCACCGATATTATTTGCCCGATGTACGCGCGCGTGGATGAAGACCAGCCCTTCCCGGCTGTGCCGAAATGGTCCATCAAAAAATGGCTTTCGCTACCTGGAGAGACGCGCCCGCTGATCCTTTGCGAATACGCCCACGCGATGGGTAACAGTCTTGGCGGTTTCGCTAAATACTGGCAGGCGTTTCGTCAGTATCCCCGTTTACAGGGCGGCTTCGTCTGGGACTGGGTGGATCAGTCGCTGATTAAATATGATGAAAACGGCAACCCGTGGTCGGCTTACGGCGGTGATTTTGGCGATACGCCGAACGATCGCCAGTTCTGTATGAACGGTCTGGTCTTTGCCGACCGCACGCCGCATCCAGCGCTGACGGAAGCAAAACACCAGCAGCAGTTTTTCCAGTTCCGTTTATCCGGGCAAACCATCGAAGTGACCAGCGAATACCTGTTCCGTCATAGCGATAACGAGCTCCTGCACTGGATGGTGGCGCTGGATGGTAAGCCGCTGGCAAGCGGTGAAGTGCCTCTGGATGTCGCTCCACAAGGTAAACAGTTGATTGAACTGCCTGAACTACCGCAGCCGGAGAGCGCCGGGCAACTCTGGCTCACAGTACGCGTAGTGCAACCGAACGCGACCGCATGGTCAGAAGCCGGGCACATCAGCGCCTGGCAGCAGTGGCGTCTGGCGGAAAACCTCAGTGTGACGCTCCCCGCCGCGTCCCACGCCATCCCGCATCTGACCACCAGCGAAATGGATTTTTGCATCGAGCTGGGTAATAAGCGTTGGCAATTTAACCGCCAGTCAGGCTTTCTTTCACAGATGTGGATTGGCGATAAAAAACAACTGCTGACGCCGCTGCGCGATCAGTTCACCCGTGCACCGCTGGATAACGACATTGGCGTAAGTGAAGCGACCCGCATTGACCCTAACGCCTGGGTCGAACGCTGGAAGGCGGCGGGCCATTACCAGGCCGAAGCAGCGTTGTTGCAGTGCACGGCAGATACACTTGCTGATGCGGTGCTGATTACGACCGCTCACGCGTGGCAGCATCAGGGGAAAACCTTATTTATCAGCCGGAAAACCTACCGGATTGATGGTAGTGGTCAAATGGCGATTACCGTTGATGTTGAAGTGGCGAGCGATACACCGCATCCGGCGCGGATTGGCCTGAACTGCCAGCTGGCGCAGGTAGCAGAGCGGGTAAACTGGCTCGGATTAGGGCCGCAAGAAAACTATCCCGACCGCCTTACTGCCGCCTGTTTTGACCGCTGGGATCTGCCATTGTCAGACATGTATACCCCGTACGTCTTCCCGAGCGAAAACGGTCTGCGCTGCGGGACGCGCGAATTGAATTATGGCCCACACCAGTGGCGCGGCGACTTCCAGTTCAACATCAGCCGCTACAGTCAACAGCAACTGATGGAAACCAGCCATCGCCATCTGCTGCACGCGGAAGAAGGCACATGGCTGAATATCGACGGTTTCCATATGGGGATTGGTGGCGACGACTCCTGGAGCCCGTCAGTATCGGCGGAGTTCCAGCTGAGCGCCGGTCGCTACCATTACCAGTTGGTCTGGTGTCAAAAAACTAAGGGTGGGCGCGCCGACCCAGCTTTCTTGTACAAAGTGGTTGATCTAGAGGGCCCGCGGTTCGAAGGTAAGCCTATCCCTAACCCTCTCCTCGGTCTCGATTCTACGCGTACCGGTTAGTAATGAGTTTAAACGGGGGAGGCTAACTGAAACACGGAAGGAGACAATACCGGAAGGAACCCGCGCTATGACGGCAATAAAAAGACAGAATAAAACGCACGGGTGTTGGGTCGTTTGTTCATAAACGCGGGGTTCGGTCCCAGGGCTGGCACTCTGTCGATACCCCACCGAGACCCCATTGGGGCCAATACGCCCGCGTTTCTTCCTTTTCCCCACCCCACCCCCCAAGTTCGGGTGAAGGCCCAGGGCTCGCAGCCAACGTCGGGGCGGCAGGCCCTGCCATAGCAGATCCGATTCGACAGATCACTGAAATGTGTGGGCGTGGCTTAAGGGTGGGAAAGAATATATAAGGTGGGGGTCTTATGTAGTTTTGTATCTGTTTTGCAGCAGCCGCCGCCGCCATGAGCACCAACTCGTTTGATGGAAGCATTGTGAGCTCATATTTGACAACGCGCATGCCCCCATGGGCCGGGGTGCGTCAGAATGTGATGGGCTCCAGCATTGATGGTCGCCCCGTCCTGCCCGCAAACTCTACTACCTTGACCTACGAGACCGTGTCTGGAACGCCGTTGGAGACTGCAGCCTCCGCCGCCGCTTCAGCCGCTGCAGCCACCGCCCGCGGGATTGTGACTGACTTTGCTTTCCTGAGCCCGCTTGCAAGCAGTGCAGCTTCCCGTTCATCCGCCCGCGATGACAAGTTGACGGCTCTTTTGGCACAATTGGATTCTTTGACCCGGGAACTTAATGTCGTTTCTCAGCAGCTGTTGGATCTGCGCCAGCAGGTTTCTGCCCTGAAGGCTTCCTCCCCTCCCAATGCGGTTTAAAACATAAATAAAAAACCAGACTCTGTTTGGATTTGGATCAAGCAAGTGTCTTGCTGTCTTTATTTAGGGGTTTTGCGCGCGCGGTAGGCCCGGGACCAGCGGTCTCGGTCGTTGAGGGTCCTGTGTATTTTTTCCAGGACGTGGTAAAGGTGACTCTGGATGTTCAGATACATGGGCATAAGCCCGTCTCTGGGGTGGAGGTAGCACCACTGCAGAGCTTCATGCTGCGGGGTGGTGTTGTAGATGATCCAGTCGTAGCAGGAGCGCTGGGCGTGGTGCCTAAAAATGTCTTTCAGTAGCAAGCTGATTGCCAGGGGCAGGCCCTTGGTGTAAGTGTTTACAAAGCGGTTAAGCTGGGATGGGTGCATACGTGGGGATATGAGATGCATCTTGGACTGTATTTTTAGGTTGGCTATGTTCCCAGCCATATCCCTCCGGGGATTCATGTTGTGCAGAACCACCAGCACAGTGTATCCGGTGCACTTGGGAAATTTGTCATGTAGCTTAGAAGGAAATGCGTGGAAGAACTTGGAGACGCCCTTGTGACCTCCAAGATTTTCCATGCATTCGTCCATAATGATGGCAATGGGCCCACGGGCGGCGGCCTGGGCGAAGATATTTCTGGGATCACTAACGTCATAGTTGTGTTCCAGGATGAGATCGTCATAGGCCATTTTTACAAAGCGCGGGCGGAGGGTGCCAGACTGCGGTATAATGGTTCCATCCGGCCCAGGGGCGTAGTTACCCTCACAGATTTGCATTTCCCACGCTTTGAGTTCAGATGGGGGGATCATGTCTACCTGCGGGGCGATGAAGAAAACGGTTTCCGGGGTAGGGGAGATCAGCTGGGAAGAAAGCAGGTTCCTGAGCAGCTGCGACTTACCGCAGCCGGTGGGCCCGTAAATCACACCTATTACCGGGTGCAACTGGTAGTTAAGAGAGCTGCAGCTGCCGTCATCCCTGAGCAGGGGGGCCACTTCGTTAAGCATGTCCCTGACTCGCATGTTTTCCCTGACCAAATCCGCCAGAAGGCGCTCGCCGCCCAGCGATAGCAGTTCTTGCAAGGAAGCAAAGTTTTTCAACGGTTTGAGACCGTCCGCCGTAGGCATGCTTTTGAGCGTTTGACCAAGCAGTTCCAGGCGGTCCCACAGCTCGGTCACCTGCTCTACGGCATCTCGATCCAGCATATCTCCTCGTTTCGCGGGTTGGGGCGGCTTTCGCTGTACGGCAGTAGTCGGTGCTCGTCCAGACGGGCCAGGGTCATGTCTTTCCACGGGCGCAGGGTCCTCGTCAGCGTAGTCTGGGTCACGGTGAAGGGGTGCGCTCCGGGCTGCGCGCTGGCCAGGGTGCGCTTGAGGCTGGTCCTGCTGGTGCTGAAGCGCTGCCGGTCTTCGCCCTGCGCGTCGGCCAGGTAGCATTTGACCATGGTGTCATAGTCCAGCCCCTCCGCGGCGTGGCCCTTGGCGCGCAGCTTGCCCTTGGAGGAGGCGCCGCACGAGGGGCAGTGCAGACTTTTGAGGGCGTAGAGCTTGGGCGCGAGAAATACCGATTCCGGGGAGTAGGCATCCGCGCCGCAGGCCCCGCAGACGGTCTCGCATTCCACGAGCCAGGTGAGCTCTGGCCGTTCGGGGTCAAAAACCAGGTTTCCCCCATGCTTTTTGATGCGTTTCTTACCTCTGGTTTCCATGAGCCGGTGTCCACGCTCGGTGACGAAAAGGCTGTCCGTGTCCCCGTATACAGACTTGAGAGGCCTGTCCTCGAGCGGTGTTCCGCGGTCCTCCTCGTATAGAAACTCGGACCACTCTGAGACAAAGGCTCGCGTCCAGGCCAGCACGAAGGAGGCTAAGTGGGAGGGGTAGCGGTCGTTGTCCACTAGGGGGTCCACTCGCTCCAGGGTGTGAAGACACATGTCGCCCTCTTCGGCATCAAGGAAGGTGATTGGTTTGTAGGTGTAGGCCACGTGACCGGGTGTTCCTGAAGGGGGGCTATAAAAGGGGGTGGGGGCGCGTTCGTCCTCACTCTCTTCCGCATCGCTGTCTGCGAGGGCCAGCTGTTGGGGTGAGTACTCCCTCTGAAAAGCGGGCATGACTTCTGCGCTAAGATTGTCAGTTTCCAAAAACGAGGAGGATTTGATATTCACCTGGCCCGCGGTGATGCCTTTGAGGGTGGCCGCATCCATCTGGTCAGAAAAGACAATCTTTTTGTTGTCAAGCTTGGTGGCAAACGACCCGTAGAGGGCGTTGGACAGCAACTTGGCGATGGAGCGCAGGGTTTGGTTTTTGTCGCGATCGGCGCGCTCCTTGGCCGCGATGTTTAGCTGCACGTATTCGCGCGCAACGCACCGCCATTCGGGAAAGACGGTGGTGCGCTCGTCGGGCACCAGGTGCACGCGCCAACCGCGGTTGTGCAGGGTGACAAGGTCAACGCTGGTGGCTACCTCTCCGCGTAGGCGCTCGTTGGTCCAGCAGAGGCGGCCGCCCTTGCGCGAGCAGAATGGCGGTAGGGGGTCTAGCTGCGTCTCGTCCGGGGGGTCTGCGTCCACGGTAAAGACCCCGGGCAGCAGGCGCGCGTCGAAGTAGTCTATCTTGCATCCTTGCAAGTCTAGCGCCTGCTGCCATGCGCGGGCGGCAAGCGCGCGCTCGTATGGGTTGAGTGGGGGACCCCATGGCATGGGGTGGGTGAGCGCGGAGGCGTACATGCCGCAAATGTCGTAAACGTAGAGGGGCTCTCTGAGTATTCCAAGATATGTAGGGTAGCATCTTCCACCGCGGATGCTGGCGCGCACGTAATCGTATAGTTCGTGCGAGGGAGCGAGGAGGTCGGGACCGAGGTTGCTACGGGCGGGCTGCTCTGCTCGGAAGACTATCTGCCTGAAGATGGCATGTGAGTTGGATGATATGGTTGGACGCTGGAAGACGTTGAAGCTGGCGTCTGTGAGACCTACCGCGTCACGCACGAAGGAGGCGTAGGAGTCGCGCAGCTTGTTGACCAGCTCGGCGGTGACCTGCACGTCTAGGGCGCAGTAGTCCAGGGTTTCCTTGATGATGTCATACTTATCCTGTCCCTTTTTTTTCCACAGCTCGCGGTTGAGGACAAACTCTTCGCGGTCTTTCCAGTACTCTTGGATCGGAAACCCGTCGGCCTCCGAACGGTAAGAGCCTAGCATGTAGAACTGGTTGACGGCCTGGTAGGCGCAGCATCCCTTTTCTACGGGTAGCGCGTATGCCTGCGCGGCCTTCCGGAGCGAGGTGTGGGTGAGCGCAAAGGTGTCCCTGACCATGACTTTGAGGTACTGGTATTTGAAGTCAGTGTCGTCGCATCCGCCCTGCTCCCAGAGCAAAAAGTCCGTGCGCTTTTTGGAACGCGGATTTGGCAGGGCGAAGGTGACATCGTTGAAGAGTATCTTTCCCGCGCGAGGCATAAAGTTGCGTGTGATGCGGAAGGGTCCCGGCACCTCGGAACGGTTGTTAATTACCTGGGCGGCGAGCACGATCTCGTCAAAGCCGTTGATGTTGTGGCCCACAATGTAAAGTTCCAAGAAGCGCGGGATGCCCTTGATGGAAGGCAATTTTTTAAGTTCCTCGTAGGTGAGCTCTTCAGGGGAGCTGAGCCCGTGCTCTGAAAGGGCCCAGTCTGCAAGATGAGGGTTGGAAGCGACGAATGAGCTCCACAGGTCACGGGCCATTAGCATTTGCAGGTGGTCGCGAAAGGTCCTAAACTGGCGACCTATGGCCATTTTTTCTGGGGTGATGCAGTAGAAGGTAAGCGGGTCTTGTTCCCAGCGGTCCCATCCAAGGTTCGCGGCTAGGTCTCGCGCGGCAGTCACTAGAGGCTCATCTCCGCCGAACTTCATGACCAGCATGAAGGGCACGAGCTGCTTCCCAAAGGCCCCCATCCAAGTATAGGTCTCTACATCGTAGGTGACAAAGAGACGCTCGGTGCGAGGATGCGAGCCGATCGGGAAGAACTGGATCTCCCGCCACCAATTGGAGGAGTGGCTATTGATGTGGTGAAAGTAGAAGTCCCTGCGACGGGCCGAACACTCGTGCTGGCTTTTGTAAAAACGTGCGCAGTACTGGCAGCGGTGCACGGGCTGTACATCCTGCACGAGGTTGACCTGACGACCGCGCACAAGGAAGCAGAGTGGGAATTTGAGCCCCTCGCCTGGCGGGTTTGGCTGGTGGTCTTCTACTTCGGCTGCTTGTCCTTGACCGTCTGGCTGCTCGAGGGGAGTTACGGTGGATCGGACCACCACGCCGCGCGAGCCCAAAGTCCAGATGTCCGCGCGCGGCGGTCGGAGCTTGATGACAACATCGCGCAGATGGGAGCTGTCCATGGTCTGGAGCTCCCGCGGCGTCAGGTCAGGCGGGAGCTCCTGCAGGTTTACCTCGCATAGACGGGTCAGGGCGCGGGCTAGATCCAGGTGATACCTAATTTCCAGGGGCTGGTTGGTGGCGGCGTCGATGGCTTGCAAGAGGCCGCATCCCCGCGGCGCGACTACGGTACCGCGCGGCGGGCGGTGGGCCGCGGGGGTGTCCTTGGATGATGCATCTAAAAGCGGTGACGCGGGCGAGCCCCCGGAGGTAGGGGGGGCTCCGGACCCGCCGGGAGAGGGGGCAGGGGCACGTCGGCGCCGCGCGCGGGCAGGAGCTGGTGCTGCGCGCGTAGGTTGCTGGCGAACGCGACGACGCGGCGGTTGATCTCCTGAATCTGGCGCCTCTGCGTGAAGACGACGGGCCCGGTGAGCTTGAGCCTGAAAGAGAGTTCGACAGAATCAATTTCGGTGTCGTTGACGGCGGCCTGGCGCAAAATCTCCTGCACGTCTCCTGAGTTGTCTTGATAGGCGATCTCGGCCATGAACTGCTCGATCTCTTCCTCCTGGAGATCTCCGCGTCCGGCTCGCTCCACGGTGGCGGCGAGGTCGTTGGAAATGCGGGCCATGAGCTGCGAGAAGGCGTTGAGGCCTCCCTCGTTCCAGACGCGGCTGTAGACCACGCCCCCTTCGGCATCGCGGGCGCGCATGACCACCTGCGCGAGATTGAGCTCCACGTGCCGGGCGAAGACGGCGTAGTTTCGCAGGCGCTGAAAGAGGTAGTTGAGGGTGGTGGCGGTGTGTTCTGCCACGAAGAAGTACATAACCCAGCGTCGCAACGTGGATTCGTTGATATCCCCCAAGGCCTCAAGGCGCTCCATGGCCTCGTAGAAGTCCACGGCGAAGTTGAAAAACTGGGAGTTGCGCGCCGACACGGTTAACTCCTCCTCCAGAAGACGGATGAGCTCGGCGACAGTGTCGCGCACCTCGCGCTCAAAGGCTACAGGGGCCTCTTCTTCTTCTTCAATCTCCTCTTCCATAAGGGCCTCCCCTTCTTCTTCTTCTGGCGGCGGTGGGGGAGGGGGGACACGGCGGCGACGACGGCGCACCGGGAGGCGGTCGACAAAGCGCTCGATCATCTCCCCGCGGCGACGGCGCATGGTCTCGGTGACGGCGCGGCCGTTCTCGCGGGGGCGCAGTTGGAAGACGCCGCCCGTCATGTCCCGGTTATGGGTTGGCGGGGGGCTGCCATGCGGCAGGGATACGGCGCTAACGATGCATCTCAACAATTGTTGTGTAGGTACTCCGCCGCCGAGGGACCTGAGCGAGTCCGCATCGACCGGATCGGAAAACCTCTCGAGAAAGGCGTCTAACCAGTCACAGTCGCAAGGTAGGCTGAGCACCGTGGCGGGCGGCAGCGGGCGGCGGTCGGGGTTGTTTCTGGCGGAGGTGCTGCTGATGATGTAATTAAAGTAGGCGGTCTTGAGACGGCGGATGGTCGACAGAAGCACCATGTCCTTGGGTCCGGCCTGCTGAATGCGCAGGCGGTCGGCCATGCCCCAGGCTTCGTTTTGACATCGGCGCAGGTCTTTGTAGTAGTCTTGCATGAGCCTTTCTACCGGCACTTCTTCTTCTCCTTCCTCTTGTCCTGCATCTCTTGCATCTATCGCTGCGGCGGCGGCGGAGTTTGGCCGTAGGTGGCGCCCTCTTCCTCCCATGCGTGTGACCCCGAAGCCCCTCATCGGCTGAAGCAGGGCTAGGTCGGCGACAACGCGCTCGGCTAATATGGCCTGCTGCACCTGCGTGAGGGTAGACTGGAAGTCATCCATGTCCACAAAGCGGTGGTATGCGCCCGTGTTGATGGTGTAAGTGCAGTTGGCCATAACGGACCAGTTAACGGTCTGGTGACCCGGCTGCGAGAGCTCGGTGTACCTGAGACGCGAGTAAGCCCTCGAGTCAAATACGTAGTCGTTGCAAGTCCGCACCAGGTACTGGTATCCCACCAAAAAGTGCGGCGGCGGCTGGCGGTAGAGGGGCCAGCGTAGGGTGGCCGGGGCTCCGGGGGCGAGATCTTCCAACATAAGGCGATGATATCCGTAGATGTACCTGGACATCCAGGTGATGCCGGCGGCGGTGGTGGAGGCGCGCGGAAAGTCGCGGACGCGGTTCCAGATGTTGCGCAGCGGCAAAAAGTGCTCCATGGTCGGGACGCTCTGGCCGGTCAGGCGCGCGCAATCGTTGACGCTCTAGACCGTGCAAAAGGAGAGCCTGTAAGCGGGCACTCTTCCGTGGTCTGGTGGATAAATTCGCAAGGGTATCATGGCGGACGACCGGGGTTCGAGCCCCGTATCCGGCCGTCCGCCGTGATCCATGCGGTTACCGCCCGCGTGTCGAACCCAGGTGTGCGACGTCAGACAACGGGGGAGTGCTCCTTTTGGCTTCCTTCCAGGCGCGGCGGCTGCTGCGCTAGCTTTTTTGGCCACTGGCCGCGCGCAGCGTAAGCGGTTAGGCTGGAAAGCGAAAGCATTAAGTGGCTCGCTCCCTGTAGCCGGAGGGTTATTTTCCAAGGGTTGAGTCGCGGGACCCCCGGTTCGAGTCTCGGACCGGCCGGACTGCGGCGAACGGGGGTTTGCCTCCCCGTCATGCAAGACCCCGCTTGCAAATTCCTCCGGAAACAGGGACGAGCCCCTTTTTTGCTTTTCCCAGATGCATCCGGTGCTGCGGCAGATGCGCCCCCCTCCTCAGCAGCGGCAAGACCAAGAGCAGCGGCAGACATGCAGGGCACCCTCCCCTCCTCCTACCGCGTCAGGAGGGGCGACATCCGCGGTTGACGCGGCAGCAGATGGTGATTACGAACCCCCGCGGCGCCGGGCCCGGCACTACCTGGACTTGGAGGAGGGCGAGGGCCTGGCGCGGCTAGGAGCGCCCTCTCCTGAGCGGTACCCAAGGGTGCAGCTGAAGCGTGATACGCGTGAGGCGTACGTGCCGCGGCAGAACCTGTTTCGCGACCGCGAGGGAGAGGAGCCCGAGGAGATGCGGGATCGAAAGTTCCACGCAGGGCGCGAGCTGCGGCATGGCCTGAATCGCGAGCGGTTGCTGCGCGAGGAGGACTTTGAGCCCGACGCGCGAACCGGGATTAGTCCCGCGCGCGCACACGTGGCGGCCGCCGACCTGGTAACCGCATACGAGCAGACGGTGAACCAGGAGATTAACTTTCAAAAAAGCTTTAACAACCACGTGCGTACGCTTGTGGCGCGCGAGGAGGTGGCTATAGGACTGATGCATCTGTGGGACTTTGTAAGCGCGCTGGAGCAAAACCCAAATAGCAAGCCGCTCATGGCGCAGCTGTTCCTTATAGTGCAGCACAGCAGGGACAACGAGGCATTCAGGGATGCGCTGCTAAACATAGTAGAGCCCGAGGGCCGCTGGCTGCTCGATTTGATAAACATCCTGCAGAGCATAGTGGTGCAGGAGCGCAGCTTGAGCCTGGCTGACAAGGTGGCCGCCATCAACTATTCCATGCTTAGCCTGGGCAAGTTTTACGCCCGCAAGATATACCATACCCCTTACGTTCCCATAGACAAGGAGGTAAAGATCGAGGGGTTCTACATGCGCATGGCGCTGAAGGTGCTTACCTTGAGCGACGACCTGGGCGTTTATCGCAACGAGCGCATCCACAAGGCCGTGAGCGTGAGCCGGCGGCGCGAGCTCAGCGACCGCGAGCTGATGCACAGCCTGCAAAGGGCCCTGGCTGGCACGGGCAGCGGCGATAGAGAGGCCGAGTCCTACTTTGACGCGGGCGCTGACCTGCGCTGGGCCCCAAGCCGACGCGCCCTGGAGGCAGCTGGGGCCGGACCTGGGCTGGCGGTGGCACCCGCGCGCGCTGGCAACGTCGGCGGCGTGGAGGAATATGACGAGGACGATGAGTACGAGCCAGAGGACGGCGAGTACTAAGCGGTGATGTTTCTGATCAGATGATGCAAGACGCAACGGACCCGGCGGTGCGGGCGGCGCTGCAGAGCCAGCCGTCCGGCCTTAACTCCACGGACGACTGGCGCCAGGTCATGGACCGCATCATGTCGCTGACTGCGCGCAATCCTGACGCGTTCCGGCAGCAGCCGCAGGCCAACCGGCTCTCCGCAATTCTGGAAGCGGTGGTCCCGGCGCGCGCAAACCCCACGCACGAGAAGGTGCTGGCGATCGTAAACGCGCTGGCCGAAPACAGGGCCATCCGGCCCGACGAGGCCGGCCTGGTCTACGACGCGCTGCTTCAGCGCGTGGCTCGTTACAACAGCGGCAACGTGCAGACCAACCTGGACCGGCTGGTGGGGGATGTGCGCGAGGCCGTGGCGCAGCGTGAGCGCGCGCAGCAGCAGGGCAACCTGGGCTCCATGGTTGCACTAAACGCCTTCCTGAGTACACAGCCCGCCAACGTGCCGCGGGGACAGGAGGACTACACCAACTTTGTGAGCGCACTGCGGCTAATGGTGACTGAGACACCGCAAAGTGAGGTGTACCAGTCTGGGCCAGACTATTTTTTCCAGACCAGTAGACAAGGCCTGCAGACCGTAAACCTGAGCCAGGCTTTCAAAAACTTGCAGGGGCTGTGGGGGGTGCGGGCTCCCACAGGCGACCGCGCGACCGTGTCTAGCTTGCTGACGCCCAACTCGCGCCTGTTGCTGCTGCTAATAGCGCCCTTCACGGACAGTGGCAGCGTGTCCCGGGACACATACCTAGGTCACTTGCTGACACTGTACCGCGAGGCCATAGGTCAGGCGCATGTGGACGAGCATACTTTCCAGGAGATTACAAGTGTCAGCCGCGCGCTGGGGCAGGAGGACACGGGCAGCCTGGAGGCAACCCTAAACTACCTGCTGACCAACCGGCGGCAGAAGATCCCCTCGTTGCACAGTTTAAACAGCGAGGAGGAGCGCATTTTGCGCTACGTGCAGCAGAGCGTGAGCCTTAACCTGATGCGCGACGGGGTAACGCCCAGCGTGGCGCTGGACATGACCGCGCGCAACATGGAACCGGGCATGTATGCCTCAAACCGGCCGTTTATCAACCGCCTAATGGACTACTTGCATCGCGCGGCCGCCGTGAACCCCGAGTATTTCACCAATGCCATCTTGAACCCGCACTGGCTACCGCCCCCTGGTTTCTACACCGGGGGATTCGAGGTGCCCGAGGGTAACGATGGATTCCTCTGGGACGACATAGACGACAGCGTGTTTTCCCCGCAACCGCAGACCCTGCTAGAGTTGCAACAGCGCGAGCAGGCAGAGGCGGCGCTGCGAAAGGAAAGCTTCCGCAGGCCAAGCAGCTTGTCCGATCTAGGCGCTGCGGCCCCGCGGTCAGATGCTAGTAGCCCATTTCCAAGCTTGATAGGGTCTCTTACCAGCACTCGCACCACCCGCCCGCGCCTGCTGGGCGAGGAGGAGTACCTAAACAACTCGCTGCTGCAGCCGCAGCGCGAAAAAAACCTGCCTCCGGCATTTCCCAACAACGGGATAGAGAGCCTAGTGGACAAGATGAGTAGATGGAAGACGTACGCGCAGGAGCACAGGGACGTGCCAGGCCCGCGCCCGCCCACCCGTCGTCAAAGGCACGACCGTCAGCGGGGTCTGGTGTGGGAGGACGATGACTCGGCAGACGACAGCAGCGTCCTGGATTTGGGAGGGAGTGGCAACCCGTTTGCGCACCTTCGCCCCAGGCTGGGGAGAATGTTTTAAAAAAAAAAAAGCATGATGCAAAATAAAAAACTCACCAAGGCCATGGCACCGAGCGTTGGTTTTCTTGTATTCCCCTTAGTATGCGGCGCGCGGCGATGTATGAGGAAGGTCCTCCTCCCTCCTACGAGAGTGTGGTGAGCGCGGCGCCAGTGGCGGCGGCGCTGGGTTCTCCCTTCGATGCTCCCCTGGACCCGCCGTTTGTGCCTCCGCGGTACCTGCGGCCTACCGGGGGGAGAAACAGCATCCGTTACTCTGAGTTGGCACCCCTATTCGACACCACCCGTGTGTACCTGGTGGACAACAAGTCAACGGATGTGGCATCCCTGAACTACCAGAACGACCACAGCAACTTTCTGACCACGGTCATTCAAAACAATGACTACAGCCCGGGGGAGGCAAGCACACAGACCATCAATCTTGACGACCGGTCGCACTGGGGCGGCGACCTGAAAACCATCCTGCATACCAACATGCCAAATGTGAACGAGTTCATGTTTACCAATAAGTTTAAGGCGCGGGTGATGGTGTCGCGCTTGCCTACTAAGGACAATCAGGTGGAGCTGAAATACGAGTGGGTGGAGTTCACGCTGCCCGAGGGCAACTACTCCGAGACCATGACCATAGACCTTATGAACAACGCGATCGTGGAGCACTACTTGAAAGTGGGCAGACAGAACGGGGTTCTGGAAAGCGACATCGGGGTAAAGTTTGACACCCGCAACTTCAGACTGGGGTTTGACCCCGTCACTGGTCTTGTCATGCCTGGGGTATATACAAACGAAGCCTTCCATCCAGACATCATTTTGCTGCCAGGATGCGGGGTGGACTTCACCCACAGCCGCCTGAGCAACTTGTTGGGCATCCGCAAGCGGCAACCCTTCCAGGAGGGCTTTAGGATCACCTACGATGATCTGGAGGGTGGTAACATTCCCGCACTGTTGGATGTGGACGCCTACCAGGCGAGCTTGAAAGATGACACCGAACAGGGCGGGGGTGGCGCAGGCGGCAGCAACAGCAGTGGCAGCGGCGCGGAAGAGAACTCCAACGCGGCAGCCGCGGCAATGCAGCCGGTGGAGGACATGAACGATCATGCCATTCGCGGCGACACCTTTGCCACACGGGCTGAGGAGAAGCGCGCTGAGGCCGAAGCAGCGGCCGAAGCTGCCGCCCCCGCTGCGCAACCCGAGGTCGAGAAGCCTCAGAAGAAACCGGTGATCAAACCCCTGACAGAGGACAGCAAGAAACGCAGTTACAACCTAATAAGCAATGACAGCACCTTCACCCAGTACCGCAGCTGGTACCTTGCATACAACTACGGCGACCCTCAGACCGGAATCCGCTCATGGACCCTGCTTTGCACTCCTGACGTAACCTGCGGCTCGGAGCAGGTCTACTGGTCGTTGCCAGACATGATGCAAGACCCCGTGACCTTCCGCTCCACGCGCCAGATCAGCAACTTTCCGGTGGTGGGCGCCGAGCTGTTGCCCGTGCACTCCAAGAGCTTCTACAACGACCAGGCCGTCTACTCCCAACTCATCCGCCAGTTTACCTCTCTGACCCACGTGTTCAATCGCTTTCCCGAGAACCAGATTTTGGCGCGCCCGCCAGCCCCCACCATCACCACCGTCAGTGAAAACGTTCCTGCTCTCACAGATCACGGGACGCTACCGCTGCGCAACAGCATCGGAGGAGTCCAGCGAGTGACCATTACTGACGCCAGACGCCGCACCTGCCCCTACGTTTACAAGGCCCTGGGCATAGTCTCGCCGCGCGTCCTATCGAGCCGCACTTTTTGAGCAAGCATGTCCATCCTTATATCGCCCAGCAATAACACAGGCTGGGGCCTGCGCTTCCCAAGCAAGATGTTTGGCGGGGCCAAGAAGCGCTCCGACCAACACCCAGTGCGCGTGCGCGGGCACTACCGCGCGCCCTGGGGCGCGCACAAACGCGGCCGCACTGGGCGCACCACCGTCGATGACGCCATCGACGCGGTGGTGGAGGAGGCGCGCAACTACACGCCCACGCCGCCACCAGTGTCCACAGTGGACGCGGCCATTCAGACCGTGGTGCGCGGAGCCCGGCGCTATGCTAAAATGAAGAGACGGCGGAGGCGCGTAGCACGTCGCCACCGCCGCCGACCCGGCACTGCCGCCCAACGCGCGGCGGCGGCCCTGCTTAACCGCGCACGTCGCACCGGCCGACGGGCGGCCATGCGGGCCGCTCGAAGGCTGGCCGCGGGTATTGTCACTGTGCCCCCCAGGTCCAGGCGACGAGCGGCCGCCGCAGCAGCCGCGGCCATTAGTGCTATGACTCAGGGTCGCAGGGGCAACGTGTATTGGGTGCGCGACTCGGTTAGCGGCCTGCGCGTGCCCGTGCGCACCCGCCCCCCGCGCAACTAGATTGCAAGAAAAAACTACTTAGACTCGTACTGTTGTATGTATCCAGCGGCGGCGGCGCGCAACGAAGCTATGTCCAAGCGCAAAATCAAAGAAGAGATGCTCCAGGTCATCGCGCCGGAGATCTATGGCCCCCCGAAGAAGGAAGAGCAGGATTACAAGCCCCGAAAGCTAAAGCGGGTCAAAAAGAAAAAGAAAGATGATGATGATGAACTTGACGACGAGGTGGAACTGCTGCACGCTACCGCGCCCAGGCGACGGGTACAGTGGAAAGGTCGACGCGTAAAACGTGTTTTGCGACCCGGCACCACCGTAGTCTTTACGCCCGGTGAGCGCTCCACCCGCACCTACAAGCGCGTGTATGATGAGGTGTACGGCGACGAGGACCTGCTTGAGCAGGCCAACGAGCGCCTCGGGGAGTTTGCCTACGGAAAGCGGCATAAGGACATGCTGGCGTTGCCGCTGGACGAGGGCAACCCAACACCTAGCCTAAAGCCCGTAACACTGCAGCAGGTGCTGCCCGCGCTTGCACCGTCCGAAGAAAAGCGCGGCCTAAAGCGCGAGTCTGGTGACTTGGCACCCACCGTGCAGCTGATGGTACCCAAGCGCCAGCGACTGGAAGATGTCTTGGAAAAAATGACCGTGGAACCTGGGCTGGAGCCCGAGGTCCGCGTGCGGCCAATCAAGCAGGTGGCGCCGGGACTGGGCGTGCAGACCGTGGACGTTCAGATACCCACTACCAGTAGCACCAGTATTGCCACCGCCACAGAGGGCATGGAGACACAAACGTCCCCGGTTGCCTCAGCGGTGGCGGATGCCGCGGTGCAGGCGGTCGCTGCGGCCGCGTCCAAGACCTCTACGGAGGTGCAAACGGACCCGTGGATGTTTCGCGTTTCAGCCCCCCGGCGCCCGCGCGGTTCGAGGAAGTACGGCGCCGCCAGCGCGCTACTGCCCGAATATGCCCTACATCCTTCCATTGCGCCTACCCCCGGCTATCGTGGCTACACCTACCGCCCCAGAAGACGAGCAACTACCCGACGCCGAACCACCACTGGAACCCGCCGCCGCCGTCGCCGTCGCCAGCCCGTGCTGGCCCCGATTTCCGTGCGCAGGGTGGCTCGCGAAGGAGGCAGGACCCTGGTGCTGCCAACAGCGCGCTACCACCCCAGCATCGTTTAAAAGCCGGTCTTTGTGGTTCTTGCAGATATGGCCCTCACCTGCCGCCTCCGTTTCCCGGTGCCGGGATTCCGAGGAAGAATGCACCGTAGGAGGGGCATGGCCGGCCACGGCCTGACGGGCGGCATGCGTCGTGCGCACCACCGGCGGCGGCGCGCGTCGCACCGTCGCATGCGCGGCGGTATCCTGCCCCTCCTTATTCCACTGATCGCCGCGGCGATTGGCGCCGTGCCCGGAATTGCATCCGTGGCCTTGCAGGCGCAGAGACACTGATTAAAAACAAGTTGCATGTGGAAAAATCAAAATAAAAAGTCTGGACTCTCACGCTCGCTTGGTCCTGTAACTATTTTGTAGAATGGAAGACATCAACTTTGCGTCTCTGGCCCCGCGACACGGCTCGCGCCCGTTCATGGGAAACTGGCAAGATATCGGCACCAGCAATATGAGCGGTGGCGCCTTCAGCTGGGGCTCGCTGTGGAGCGGCATTAAAAATTTCGGTTCCACCGTTAAGAACTATGGCAGCAAGGCCTGGAACAGCAGCACAGGCCAGATGCTGAGGGATAAGTTGAAAGAGCAAAATTTCCAACAAAAGGTGGTAGATGGCCTGGCCTCTGGCATTAGCGGGGTGGTGGACCTGGCCAACCAGGCAGTGCAAAATAAGATTAACAGTAAGCTTGATCCCCGCCCTCCCGTAGAGGAGCCTCCACCGGCCGTGGAGACAGTGTCTCCAGAGGGGCGTGGCGAAAAGCGTCCGCGCCCCGACAGGGAAGAAACTCTGGTGACGCAAATAGACGAGCCTCCCTCGTACGAGGAGGCACTAAAGCAAGGCCTGCCCACCACCCGTCCCATCGCGCCCATGGCTACCGGAGTGCTGGGCCAGCACACACCCGTAACGCTGGACCTGCCTCCCCCCGCCGACACCCAGCAGAAACCTGTGCTGCCAGGCCCGACCGCCGTTGTTGTAACCCGTCCTAGCCGCGCGTCCCTGCGCCGCGCCGCCAGCGGTCCGCGATCGTTGCGGCCCGTAGCCAGTGGCAACTGGCAAAGCACACTGAACAGCATCGTGGGTCTGGGGGTGCAATCCCTGAAGCGCCGACGATGCTTCTGAATAGCTAACGTGTCGTATGTGTGTCATGTATGCGTCCATGTCGCCGCCAGAGGAGCTGCTGAGCCGCCGCGCGCCCGCTTTCCAAGATGGCTACCCCTTCGATGATGCCGCAGTGGTCTTACATGCACATCTCGGGCCAGGACGCCTCGGAGTACCTGAGCCCCGGGCTGGTGCAGTTTGCCCGCGCCACCGAGACGTACTTCAGCCTGAATAACAAGTTTAGAAACCCCACGGTGGCGCCTACGCACGACGTGACCACAGACCGGTCCCAGCGTTTGACGCTGCGGTTCATCCCTGTGGACCGTGAGGATACTGCGTACTCGTACAAGGCGCGGTTCACCCTAGCTGTGGGTGATAACCGTGTGCTGGACATGGCTTCCACGTACTTTGACATCCGCGGCGTGCTGGACAGGGGCCCTACTTTTAAGCCCTACTCTGGCACTGCCTACAACGCCCTGGCTCCCAAGGGTGCCCCAAATCCTTGCGAATGGGATGAAGCTGCTACTGCTCTTGAAATAAACCTAGAAGAAGAGGACGATGACAACGAAGACGAAGTAGACGAGCAAGCTGAGCAGCAAAAAACTCACGTATTTGGGCAGGCGCCTTATTCTGGTATAAATATTACAAAGGAGGGTATTCAAATAGGTGTCGAAGGTCAAACACCTAAATATGCCGATAAAACATTTCAACCTGAACCTCAAATAGGAGAATCTCAGTGGTACGAAACTGAAATTAATCATGCAGCTGGGAGAGTCCTTAAAAAGACTACCCCAATGAAACCATGTTACGGTTCATATGCAAAACCCACAAATGAAAATGGAGGGCAAGGCATTCTTGTAAAGCAACAAAATGGAAAGCTAGAAAGTCAAGTGGAAATGCAATTTTTCTCAACTACTGAGGCGACCGCAGGCAATGGTGATAACTTGACTCCTAAAGTGGTATTGTACAGTGAAGATGTAGATATAGAAACCCCAGACACTCATATTTCTTACATGCCCACTATTAAGGAAGGTAACTCACGAGAACTAATGGGCCAACAATCTATGCCCAACAGGCCTAATTACATTGCTTTTAGGGACAATTTTATTGGTCTAATGTATTACAACAGCACGGGTAATATGGGTGTTCTGGCGGGCCAAGCATCGCAGTTGAATGCTGTTGTAGATTTGCAAGACAGAAACACAGAGCTTTCATACCAGCTTTTGCTTGATTCCATTGGTGATAGAACCAGGTACTTTTCTATGTGGAATCAGGCTGTTGACAGCTATGATCCAGATGTTAGAATTATTGAAAATCATGGAACTGAAGATGAACTTCCAAATTACTGCTTTCCACTGGGAGGTGTGATTAATACAGAGACTCTTACCAAGGTAAAACCTAAAACAGGTCAGGAAAATGGATGGGAAAAAGATGCTACAGAATTTTCAGATAAAAATGAAATAAGAGTTGGAAATAATTTTGCCATGGAAATCAATCTAAATGCCAACCTGTGGAGAAATTTCCTGTACTCCAACATAGCGCTGTATTTGCCCGACAAGCTAAAGTACAGTCCTTCCAACGTAAAAATTTCTGATAACCCAAACACCTACGACTACATGAACAAGCGAGTGGTGGCTCCCGGGTTAGTGGACTGCTACATTAACCTTGGAGCACGCTGGTCCCTTGACTATATGGACAACGTCAACCCATTTAACCACCACCGCAATGCTGGCCTGCGCTACCGCTCAATGTTGCTGGGCAATGGTCGCTATGTGCCCTTCCACATCCAGGTGCCTCAGAAGTTCTTTGCCATTAAAAACCTCCTTCTCCTGCCGGGCTCATACACCTACGAGTGGAACTTCAGGAAGGATGTTAACATGGTTCTGCAGAGCTCCCTAGGAAATGACCTAAGGGTTGACGGAGCCAGCATTAAGTTTGATAGCATTTGCCTTTACGCCACCTTCTTCCCCATGGCCCACAACACCGCCTCCACGCTTGAGGCCATGCTTAGAAACGACACCAACGACCAGTCCTTTAACGACTATCTCTCCGCCGCCAACATGCTCTACCCTATACCCGCCAACGCTACCAACGTGCCCATATCCATCCCCTCCCGCAACTGGGCGGCTTTCCGCGGCTGGGCCTTCACGCGCCTTAAGACTAAGGAAACCCCATCACTGGGCTCGGGCTACGACCCTTATTACACCTACTCTGGCTCTATACCCTACCTAGATGGAACCTTTTACCTCAACCACACCTTTAAGAAGGTGGCCATTACCTTTGACTCTTCTGTCAGCTGGCCTGGCAATGACCGCCTGCTTACCCCCAACGAGTTTGAAATTAAGCGCTCAGTTGACGGGGAGGGTTACAACGTTGCCCAGTGTAACATGACCAAAGACTGGTTCCTGGTACAAATGCTAGCTAACTACAACATTGGCTACCAGGGCTTCTATATCCCAGAGAGCTACAAGGACCGCATGTACTCCTTCTTTAGAAACTTCCAGCCCATGAGCCGTCAGGTGGTGGATGATACTAAATACAAGGACTACCAACAGGTGGGCATCCTACACCAACACAACAACTCTGGATTTGTTGGCTACCTTGCCCCCACCATGCGCGAAGGACAGGCCTACCCTGCTAACTTCCCCTATCCGCTTATAGGCAAGACCGCAGTTGACAGCATTACCCAGAAAAAGTTTCTTTGCGATCGCACCCTTTGGCGCATCCCATTCTCCAGTAACTTTATGTCCATGGGCGCACTCACAGACCTGGGCCAAAACCTTCTCTACGCCAACTCCGCCCACGCGCTAGACATGACTTTTGAGGTGGATCCCATGGACGAGCCCACCCTTCTTTATGTTTTGTTTGAAGTCTTTGACGTGGTCCGTGTGCACCGGCCGCACCGCGGCGTCATCGAAACCGTGTACCTGCGCACGCCCTTCTCGGCCGGCAACGCCACAACATAAAGAAGCAAGCAACATCAACAACAGCTGCCGCCATGGGCTCCAGTGAGCAGGAACTGAAAGCCATTGTCAAAGATCTTGGTTCTGGGCCATATTTTTTGGGCACCTATGACAAGCGCTTTCCAGGCTTTGTTTCTCCACACAAGCTCGCCTGCGCCATAGTCAATACGGCCGGTCGCGAGACTGGGGGCGTACACTGGATGGCCTTTGCCTGGAACCCGCACTCAAAAACATGCTACCTCTTTGAGCCCTTTGGCTTTTCTGACCAGCGACTCAAGCAGGTTTACCAGTTTGAGTACGAGTCACTCCTGCGCCGTAGCGCCATTGCTTCTTCCCCCGACCGCTGTATAACGCTGGAAAAGTCCACCCAAAGCGTACAGGGGCCCAACTCGGCCGCCTGTGGACTATTCTGCTGCATGTTTCTCCACGCCTTTGCCAACTGGCCCCAAACTCCCATGGATCACAACCCCACCATGAACCTTATTACCGGGGTACCCAACTCCATGCTCAACAGTCCCCAGGTACAGCCCACCCTGCGTCGCAACCAGGAACAGCTCTACAGCTTCCTGGAGCGCCACTCGCCCTACTTCCGCAGCCACAGTGCGCAGATTAGGAGCGCCACTTCTTTTTGTCACTTGAAAAACATGTAAAAATAATGTACTAGAGACACTTTCAATAAAGGCAAATGCTTTTATTTGTACACTCTCGGGTGATTATTTACCCCCACCCTTGCCGTCTGCGCCGTTTAAAAATCAAAGGGGTTCTGCCGCGCATCGCTATGCGCCACTGGCAGGGACACGTTGCGATACTGGTGTTTAGTGCTCCACTTAAACTCAGGGACPACCATCCGCGGCAGCTCGGTGAAGTTTTCACTCCACAGGCTGCGCACCATCACCAACGCGTTTAGCAGGTCGGGCGCCGATATCTTGAAGTCGCAGTTGGGGCCTCCGCCCTGCGCGCGCGAGTTGCGATACACAGGGTTGCAGCACTGGAACACTATCAGCGCCGGGTGGTGCACGCTGGCCAGCACGCTCTTGTCGGAGATCAGATCCGCGTCCAGGTCCTCCGCGTTGCTCAGGGCGAACGGAGTCAACTTTGGTAGCTGCCTTCCCAAAAAGGGCGCGTGCCCAGGCTTTGAGTTGCACTCGCACCGTAGTGGCATCAAAAGGTGACCGTGCCCGGTCTGGGCGTTAGGATACAGCGCCTGCATAAAAGCCTTGATCTGCTTAAAAGCCACCTGAGCCTTTGCGCCTTCAGAGAAGAACATGCCGCAAGACTTGCCGGAAAACTGATTGGCCGGACAGGCCGCGTCGTGCACGCAGCACCTTGCGTCGGTGTTGGAGATCTGCACCACATTTCGGCCCCACCGGTTCTTCACGATCTTGGCCTTGCTAGACTGCTCCTTCAGCGCGCGCTGCCCGTTTTCGCTCGTCACATCCATTTCAATCACGTGCTCCTTATTTATCATAATGCTTCCGTGTAGACACTTAAGCTCGCCTTCGATCTCAGCGCAGCGGTGCAGCCACAACGCGCAGCCCGTGGGCTCGTGATGCTTGTAGGTCACCTCTGCAAACGACTGCAGGTACGCCTGCAGGAATCGCCCCATCATCGTCACAAAGGTCTTGTTGCTGGTGAAGGTCAGCTGCAACCCGCGGTGCTCCTCGTTCAGCCAGGTCTTGCATACGGCCGCCAGAGCTTCCACTTGGTCAGGCAGTAGTTTGAAGTTCGCCTTTAGATCGTTATCCACGTGGTACTTGTCCATCAGCGCGCGCGCAGCCTCCATGCCCTTCTCCCACGCAGACACGATCGGCACACTCAGCGGGTTCATCACCGTAATTTCACTTTCCGCTTCGCTGGGCTCTTCCTCTTCCTCTTGCGTCCGCATACCACGCGCCACTGGGTCGTCTTCATTCAGCCGCCGCACTGTGCGCTTACCTCCTTTGCCATGCTTGATTAGCACCGGTGGGTTGCTGAAACCCACCATTTGTAGCGCCACATCTTCTCTTTCTTCCTCGCTGTCCACGATTACCTCTGGTGATGGCGGGCGCTCGGGCTTGGGAGAAGGGCGCTTCTTTTTCTTCTTGGGCGCAATGGCCAAATCCGCCGCCGAGGTCGATGGCCGCGGGCTGGGTGTGCGCGGCACCAGCGCGTCTTGTGATGAGTCTTCCTCGTCCTCGGACTCGATACGCCGCCTCATCCGCTTTTTTGGGGGCGCCCGGGGAGGCGGCGGCGACGGGGACGGGGACGACACGTCCTCCATGGTTGGGGGACGTCGCGCCGCACCGCGTCCGCGCTCGGGGGTGGTTTCGCGCTGCTCCTCTTCCCGACTGGCCATTTCCTTCTCCTATAGGCAGAAAAAGATCATGGAGTCAGTCGAGAAGAAGGACAGCCTAACCGCCCCCTCTGAGTTCGCCACCACCGCCTCCACCGATGCCGCCAACGCGCCTACCACCTTCCCCGTCGAGGCACCCCCGCTTGAGGAGGAGGAAGTGATTATCGAGCAGGACCCAGGTTTTGTAAGCGAAGACGACGAGGACCGCTCAGTACCAACAGAGGATAAAAAGCAAGACCAGGACAACGCAGAGGCAAACGAGGAACAAGTCGGGCGGGGGGACGAAAGGCATGGCGACTACCTAGATGTGGGAGACGACGTGCTGTTGAAGCATCTGCAGCGCCAGTGCGCCATTATCTGCGACGCGTTGCAAGAGCGCAGCGATGTGCCCCTCGCCATAGCGGATGTCAGCCTTGCCTACGAACGCCACCTATTCTCACCGCGCGTACCCCCCAAACGCCAAGAkAACGGCACATGCGAGCCCAACCCGCGCCTCAACTTCTACCCCGTATTTGCCGTGCCAGAGGTGCTTGCCACCTATCACATCTTTTTCCAAAACTGCAAGATACCCCTATCCTGCCGTGCCAACCGCAGCCGAGCGGACAAGCAGCTGGCCTTGCGGCAGGGCGCTGTCATACCTGATATCGCCTCGCTCAACGAAGTGCCAAAAATCTTTGAGGGTCTTGGACGCGACGAGAAGCGCGCGGCAAACGCTCTGCAACAGGAAAACAGCGAAAATGAAAGTCACTCTGGAGTGTTGGTGGAACTCGAGGGTGACAACGCGCGCCTAGCCGTACTAAAACGCAGCATCGAGGTCACCCACTTTGCCTACCCGGCACTTAACCTACCCCCCAAGGTCATGAGCACAGTCATGAGTGAGCTGATCGTGCGCCGTGCGCAGCCCCTGGAGAGGGATGCAAATTTGCAAGAACAAACAGAGGAGGGCCTACCCGCAGTTGGCGACGAGCAGCTAGCGCGCTGGCTTCAAACGCGCGAGCCTGCCGACTTGGAGGAGCGACGCAAACTAATGATGGCCGCAGTGCTCGTTACCGTGGAGCTTGAGTGCATGCAGCGGTTCTTTGCTGACCCGGAGATGCAGCGCAAGCTAGAGGAAACATTGCACTACACCTTTCGACAGGGCTACGTACGCCAGGCCTGCAAGATCTCCAACGTGGAGCTCTGCAACCTGGTCTCCTACCTTGGAATTTTGCACGAAAACCGCCTTGGGCAAAACGTGCTTCATTCCACGCTCAAGGGCGAGGCGCGCCGCGACTACGTCCGCGACTGCGTTTACTTATTTCTATGCTACACCTGGCAGACGGCCATGGGCGTTTGGCAGCAGTGCTTGGAGGAGTGCAACCTCAAGGAGCTGCAGAAACTGCTAAAGCAAAACTTGAAGGACCTATGGACGGCCTTCAACGAGCGCTCCGTGGCCGCGCACCTGGCGGACATCATTTTCCCCGAACGCCTGCTTAAAACCCTGCAACAGGGTCTGCCAGACTTCACCAGTCAAAGCATGTTGCAGAACTTTAGGAACTTTATCCTAGAGCGCTCAGGAATCTTGCCCGCCACCTGCTGTGCACTTCCTAGCGACTTTGTGCCCATTAAGTACCGCGAATGCCCTCCGCCGCTTTGGGGCCACTGCTACCTTCTGCAGCTAGCCAACTACCTTGCCTACCACTCTGACATAATGGAAGACGTGAGCGGTGACGGTCTACTGGAGTGTCACTGTCGCTGCAACCTATGCACCCCGCACCGCTCCCTGGTTTGCAATTCGCAGCTGCTTAACGAAAGTCAAATTATCGGTACCTTTGAGCTGCAGGGTCCCTCGCCTGACGAAAAGTCCGCGGCTCCGGGGTTGAAACTCACTCCGGGGCTGTGGACGTCGGCTTACCTTCGCAAATTTGTACCTGAGGACTACCACGCCCACGAGATTAGGTTCTACGAAGACCAATCCCGCCCGCCAAATGCGGAGCTTACCGCCTGCGTCATTACCCAGGGCCACATTCTTGGCCAATTGCAAGCCATCAACAAAGCCCGCCAAGAGTTTCTGCTACGAAAGGGACGGGGGGTTTACTTGGACCCCCAGTCCGGCGAGGAGCTCAACCCAATCCCCCCGCCGCCGCAGCCCTATCAGCAGCAGCCGCGGGCCCTTGCTTCCCAGGATGGCACCCAAAAAGAAGCTGCAGCTGCCGCCGCCACCCACGGACGAGGAGGAATACTGGGACAGTCAGGCAGAGGAGGTTTTGGACGAGGAGGAGGAGGACATGATGGAAGACTGGGAGAGCCTAGACGAGGAAGCTTCCGAGGTCGAAGAGGTGTCAGACGAAACACCGTCACCCTCGGTCGCATTCCCCTCGCCGGCGCCCCAGAAATCGGCAACCGGTTCCAGCATGGCTACAACCTCCGCTCCTCAGGCGCCGCCGGCACTGCCCGTTCGCCGACCCAACCGTAGATGGGACACCACTGGAACCAGGGCCGGTAAGTCCAAGCAGCCGCCGCCGTTAGCCCAAGAGCAACAACAGCGCCAAGGCTACCGCTCATGGCGCGGGCACAAGAACGCCATAGTTGCTTGCTTGCAAGACTGTGGGGGCAACATCTCCTTCGCCCGCCGCTTTCTTCTCTACCATCACGGCGTGGCCTTCCCCCGTAACATCCTGCATTACTACCGTCATCTCTACAGCCCATACTGCACCGGCGGCAGCGGCAGCGGCAGCAACAGCAGCGGCCACACAGAAGCAAAGGCGACCGGATAGCAAGACTCTGACAAAGCCCAAGAAATCCACAGCGGCGGCAGCAGCAGGAGGAGGAGCGCTGCGTCTGGCGCCCAACGAACCCGTATCGACCCGCGAGCTTAGAAACAGGATTTTTCCCACTCTGTATGCTATATTTCAACAGAGCAGGGGCCAAGAACAAGAGCTGAAAATAAAAAACAGGTCTCTGCGATCCCTCACCCGCAGCTGCCTGTATCACAAAAGCGAAGATCAGCTTCGGCGCACGCTGGAAGACGCGGAGGCTCTCTTCAGTAAATACTGCGCGCTGACTCTTAAGGACTAGTTTCGCGCCCTTTCTCAAATTTAAGCGCGAAAACTACGTCATCTCCAGCGGCCACACCCGGCGCCAGCACCTGTCGTCAGCGCCATTATGAGCAAGGAAATTCCCACGCCCTACATGTGGAGTTACCAGCCACAAATGGGACTTGCGGCTGGAGCTGCCCAAGACTACTCAACCCGAATAAACTACATGAGCGCGGGACCCCACATGATATCCCGGGTCAACGGAATCCGCGCCCACCGAAACCGAATTCTCTTGGAACAGGCGGCTATTACCACCACACCTCGTAATAACCTTAATCCCCGTAGTTGGCCCGCTGCCCTGGTGTACCAGGAAAGTCCCGCTCCCACCACTGTGGTACTTCCCAGAGACGCCCAGGCCGAAGTTCAGATGACTAACTCAGGGGCGCAGCTTGCGGGCGGCTTTCGTCACAGGGTGCGGTCGCCCGGGCAGGGTATAACTCACCTGACAATCAGAGGGCGAGGTATTCAGCTCAACGACGAGTCGGTGAGCTCCTCGCTTGGTCTCCGTCCGGACGGGACATTTCAGATCGGCGGCGCCGGCCGTCCTTCATTCACGCCTCGTCAGGCAATCCTAACTCTGCAGACCTCGTCCTCTGAGCCGCGCTCTGGAGGCATTGGAACTCTGCAATTTATTGAGGAGTTTGTGCCATCGGTCTACTTTAACCCCTTCTCGGGACCTCCCGGCCACTATCCGGATCAATTTATTCCTAACTTTGACGCGGTAAAGGACTCGGCGGACGGCTACGACTGAATGTTAAGTGGAGAGGCAGAGCAACTGCGCCTGAAACACCTGGTCCACTGTCGCCGCCACAAGTGCTTTGCCCGCGACTCCGGTGAGTTTTGCTACTTTGAATTGCCCGAGGATCATATCGAGGGCCCGGCGCACGGCGTCCGGCTTACCGCCCAGGGAGAGCTTGCCCGTAGCCTGATTCGGGAGTTTACCCAGCGCCCCCTGCTAGTTGAGCGGGACAGGGGACCCTGTGTTCTCACTGTGATTTGCAACTGTCCTAACCTTGGATTACATCAAGATCTTTGTTGCCATCTCTGTGCTGAGTATAATAAATACAGAAATTAAAATATACTGGGGCTCCTATCGCCATCCTGTAAACGCCACCGTCTTCACCCGCCCAAGCAAACCAAGGCGAACCTTACCTGGTACTTTTAACATCTCTCCCTCTGTGATTTACAACAGTTTCAACCCAGACGGAGTGAGTCTACGAGAGAACCTCTCCGAGCTCAGCTACTCCATCAGAAAAAACACCACCCTCCTTACCTGCCGGGPACGTACGAGTGCGTCACCGGCCGCTGCACCACACCTACCGCCTGACCGTAAACCAGACTTTTTCCGGACAGACCTCAATAACTCTGTTTACCAGAACAGGAGGTGAGCTTAGAAAACCCTTAGGGTATTAGGCCAAAGGCGCAGCTACTGTGGGGTTTATGAACAATTCAAGCAACTCTACGGGCTATTCTAATTCAGGTTTCTCTAGAAATGGACGGAATTATTACAGAGCAGCGCCTGCTAGAAAGACGCAGGGCAGCGGCCGAGCAACAGCGCATGAATCAAGAGCTCCAAGACATGGTTAACTTGCACCAGTGCAAAAGGGGTATCTTTTGTCTGGTAAAGCAGGCCAAAGTCACCTACGACAGTAATACCACCGGACACCGCCTTAGCTACAAGTTGCCAACCAAGCGTCAGAAATTGGTGGTCATGGTGGGAGAAAAGCCCATTACCATAACTCAGCACTCGGTAGAAACCGAAGGCTGCATTCACTCACCTTGTCAAGGACCTGAGGATCTCTGCACCCTTATTAAGACCCTGTGCGGTCTCAAAGATCTTATTCCCTTTAACTAATAAAAAAAAATAATAAAGCATCACTTACTTAAAATCAGTTAGCAAATTTCTGTCCAGTTTATTCAGCAGCACCTCCTTGCCCTCCTCCCAGCTCTGGTATTGCAGCTTCCTCCTGGCTGCAAACTTTCTCCACAATCTAAATGGAATGTCAGTTTCCTCCTGTTCCTGTCCATCCGCACCCACTATCTTCATGTTGTTGCAGATGAAGCGCGCAAGACCGTCTGAAGATACCTTCAACCCCGTGTATCCATATGACACGGAAACCGGTCCTCCAACTGTGCCTTTTCTTACTCCTCCCTTTGTATCCCCCAATGGGTTTCAAGAGAGTCCCCCTGGGGTACTCTCTTTGCGCCTATCCGAACCTCTAGTTACCTCCAATGGCATGCTTGCGCTCAAAATGGGCAACGGCCTCTCTCTGGACGAGGCCGGCAACCTTACCTCCCAAAATGTAACCACTGTGAGCCCACCTCTCAAAAAAACCAAGTCAAACATAAACCTGGAAATATCTGCACCCCTCACAGTTACCTCAGAAGCCCTAACTGTGGCTGCCGCCGCACCTCTAATGGTCGCGGGCAACACACTCACCATGCAATCACAGGCCCCGCTAACCGTGCACGACTCCAAACTTAGCATTGCCACCCAAGGACCCCTCACAGTGTCAGAAGGAAAGCTAGCCCTGCAAACATCAGGCCCCCTCACCACCACCGATAGCAGTACCCTTACTATCACTGCCTCACCCCCTCTAACTACTGCCACTGGTAGCTTGGGCATTGACTTGAAAGAGCCCATTTATACACAAAATGGAAAACTAGGACTAAAGTACGGGGCTCCTTTGCATGTAACAGACGACCTAAACACTTTGACCGTAGCAACTGGTCCAGGTGTGACTATTAATAATACTTCCTTGCAAACTAAAGTTACTGGAGCCTTGGGTTTTGATTCACAAGGCAATATGCAACTTAATGTAGCAGGAGGACTAAGGATTGATTCTCAAAACAGACGCCTTATACTTGATGTTAGTTATCCGTTTGATGCTCAAAACCAACTAAATCTAAGACTAGGACAGGGCCCTCTTTTTATAAACTCAGCCCACAACTTGGATATTAACTACAACAAAGGCCTTTACTTGTTTACAGCTTCAAACAATTCCAAAAAGCTTGAGGTTAACCTAAGCACTGCCAAGGGGTTGATGTTTGACGCTACAGCCATAGCCATTAATGCAGGAGATGGGCTTGAATTTGGTTCACCTAATGCACCAAACACAAATCCCCTCAAAACAAAAATTGGCCATGGCCTAGAATTTGATTCAAACAAGGCTATGGTTCCTAAACTAGGAACTGGCCTTAGTTTTGACAGCACAGGTGCCATTACAGTAGGAAACAAAAATAATGATAAGCTAACTTTGTGGACCACACCAGCTCCATCTCCTAACTGTAGACTAAATGCAGAGAAAGATGCTAAACTCACTTTGGTCTTAACAAAATGTGGCAGTCAAATACTTGCTACAGTTTCAGTTTTGGCTGTTAAAGGCAGTTTGGCTCCAATATCTGGAACAGTTCAAAGTGCTCATCTTATTATAAGATTTGACGAAAATGGAGTGCTACTAAACAATTCCTTCCTGGACCCAGAATATTGGAACTTTAGAAATGGAGATCTTACTGAAGGCACAGCCTATACAAACGCTGTTGGATTTATGCCTAACCTATCAGCTTATCCAAAATCTCACGGTAAAACTGCCAAAAGTAACATTGTCAGTCAAGTTTACTTAAACGGAGACAAAACTAAACCTGTAACACTAACCATTACACTAAACGGTACACAGGAAACAGGAGACACAACTCCAAGTGCATACTCTATGTCATTTTCATGGGACTGGTCTGGCCACAACTACATTAATGAAATATTTGCCACATCCTCTTACACTTTTTCATACATTGCCCAAGAATAAAGAATCGTTTGTGTTATGTTTCAACGTGTTTATTTTTCAATTGCAGAAAATTTCGAATCATTTTTCATTCAGTAGTATAGCCCCACCACCACATAGCTTATACAGATCACCGTACCTTAATCAAACTCACAGPACCCTAGTATTCPACCTGCCACCTCCCTCCCAACACACAGAGTACACAGTCCTTTCTCCCCGGCTGGCCTTAAAAAGCATCATATCATGGGTAACAGACATATTCTTAGGTGTTATATTCCACACGGTTTCCTGTCGAGCCAAACGCTCATCAGTGATATTAATAAACTCCCCGGGCAGCTCACTTAAGTTCATGTCGCTGTCCAGCTGCTGAGCCACAGGCTGCTGTCCAACTTGCGGTTGCTTAACGGGCGGCGAAGGAGAAGTCCACGCCTACATGGGGGTAGAGTCATAATCGTGCATCAGGATAGGGCGGTGGTGCTGCAGCAGCGCGCGAATAAACTGCTGCCGCCGCCGCTCCGTCCTGCAGGAATACAACATGGCAGTGGTCTCCTCAGCGATGATTCGCACCGCCCGCAGCATAAGGCGCCTTGTCCTCCGGGCACAGCAGCGCACCCTGATCTCACTTAAATCAGCACAGTAACTGCAGCACAGCACCACAATATTGTTCAAAATCCCACAGTGCAAGGCGCTGTATCCAAAGCTCATGGCGGGGACCACAGAACCCACGTGGCCATCATACCACAAGCGCAGGTAGATTAAGTGGCGACCCCTCATAAACACGCTGGACATAAACATTACCTCTTTTGGCATGTTGTAATTCACCACCTCCCGGTACCATATAAACCTCTGATTAAACATGGCGCCATCCACCACCATCCTAAACCAGCTGGCCAAAACCTGCCCGCCGGCTATACACTGCAGGGAACCGGGACTGGAACAATGACAGTGGAGAGCCCAGGACTCGTAACCATGGATCATCATGCTCGTCATGATATCAATGTTGGCACAACACAGGCACACGTGCATACACTTCCTCAGGATTACAAGCTCCTCCCGCGTTAGAACCATATCCCAGGGAACAACCCATTCCTGAATCAGCGTAAATCCCACACTGCAGGGAAGACCTCGCACGTAACTCACGTTGTGCATTGTCAAAGTGTTACATTCGGGCAGCAGCGGATGATCCTCCAGTATGGTAGCGCGGGTTTCTGTCTCAAAAGGAGGTAGACGATCCCTACTGTACGGAGTGCGCCGAGACAACCGAGATCGTGTTGGTCGTAGTGTCATGCCAAATGGAACGCCGGACGTAGTCATATTTCCTGAAGCAAAACCAGGTGCGGGCGTGACAAACAGATCTGCGTCTCCGGTCTCGCCGCTTAGATCGCTCTGTGTAGTAGTTGTAGTATATCCACTCTCTCAAAGCATCCAGGCGCCCCCTGGCTTCGGGTTCTATGTAAACTCCTTCATGCGCCGCTGCCCTGATAACATCCACCACCGCAGAATAAGCCACACCCAGCCAACCTACACATTCGTTCTGCGAGTCACACACGGGAGGAGCGGGAAGAGCTGGAAGAACCATGTTTTTTTTTTTATTCCAAAAGATTATCCAAAACCTCAAAATGAAGATCTATTAAGTGAACGCGCTCCCCTCCGGTGGCGTGGTCAAACTCTACAGCCAAAGAACAGATAATGGCATTTGTAAGATGTTGCACAATGGCTTCCAAAAGGCAAACGGCCCTCACGTCCAAGTGGACGTAAAGGCTAAACCCTTCAGGGTGAATCTCCTCTATAAACATTCCAGCACCTTCAACCATGCCCAAATAATTCTCATCTCGCCACCTTCTCAATATATCTCTAAGCAAATCCCGAATATTAAGTCCGGCCATTGTAAAAATCTGCTCCAGAGCGCCCTCCACCTTCAGCCTCAAGCAGCGAATCATGATTGCAAAAATTCAGGTTCCTCACAGACCTGTATAAGATTCAAAAGCGGAACATTAACAAAAATACCGCGATCCCGTAGGTCCCTTCGCAGGGCCAGCTGAACATAATCGTGCAGGTCTGCACGGACCAGCGCGGCCACTTCCCCGCCAGGAACCTTGACAAAAGAACCCACACTGATTATGACACGCATACTCGGAGCTATGCTAACCAGCGTAGCCCCGATGTAAGCTTTGTTGCATGGGCGGCGATATAAAATGCAAGGTGCTGCTCAAAAAATCAGGCAPAGCCTCGCGCAAAAAAGAAAGCACATCGTAGTCATGCTCATGCAGATAAAGGCAGGTAAGCTCCGGAACCACCACAGAAAAAGACACCATTTTTCTCTCAAACATGTCTGCGGGTTTCTGCATAAACACAAAATAAAATAACAAAAAAACATTTAAACATTAGAAGCCTGTCTTACAACAGGAAAAACAACCCTTATAAGCATAAGACGGACTACGGCCATGCCGGCGTGACCGTAAAAAAACTGGTCACCGTGATTAAAAAGCACCACCGACAGCTCCTCGGTCATGTCCGGAGTCATAATGTAAGACTCGGTAAACACATCAGGTTGATTCACATCGGTCAGTGCTAAAAAGCGACCGAAATAGCCCGGGGGAATACATACCCGCAGGCGTAGAGACAACATTACAGCCCCCATAGGAGGTATAACAAAATTAATAGGAGAGAAAAACACATAAACACCTGAAAAACCCTCCTGCCTAGGCAAAATAGCACCCTCCCGCTCCAGAACAACATACAGCGCTTCCACAGCGGCAGCCATAACAGTCAGCCTTACCAGTAAAAAAGAAAACCTATTAAAAAAACACCACTCGACACGGCACCAGCTCAATCAGTCACAGTGTAAAAAAGGGCCAAGTGCAGAGCGAGTATATATAGGACTAAAAAATGACGTAACGGTTAAAGTCCACAAAAAACACCCAGAAAACCGCACGCGAACCTACGCCCAGAAACGAAAGCCAAAAAACCCACAACTTCCTCAAATCGTCACTTCCGTTTTCCCACGTTACGTCACTTCCCATTTTAAGAAAACTACAATTCCCAACACATACAAGTTACTCCGCCCTAAAACCTACGTCACCCGCCCCGTTCCCACGCCCCGCGCCACGTCACAAACTCCACCCCCTCATTATCATATTGGCTTCAATCCAAAATAAGGTATATTATTGATGATGTTAATTAATTTAAATCCGCATGCGATATCGAGCTCTCCCGGGAATTCGGATCTGCGACGCGAGGCTGGATGGCCTTCCCCATTATGATTCTTCTCGCTTCCGGCGGCATCGGGATGCCCGCGTTGCAGGCCATGCTGTCCAGGCAGGTAGATGACGACCATCAGGGACAGCTTCACGGCCAGCAAAAGGCCAGGAACCGTAAAAAGGCCGCGTTGCTGGCGTTTTTCCATAGGCTCCGCCCCCCTGACGAGCATCACAAAAATCGACGCTCAAGTCAGAGGTGGCGAAACCCGACAGGACTATAPAGATACCAGGCGTTTCCCCCTGGAAGCTCCCTCGTGCGCTCTCCTGTTCCGACCCTGCCGCTTACCGGATACCTGTCCGCCTTTCTCCCTTCGGGAAGCGTGGCGCTTTCTCAATGCTCACGCTGTAGGTATCTCAGTTCGGTGTAGGTCGTTCGCTCCAAGCTGGGCTGTGTGCACGAACCCCCCGTTCAGCCCGACCGCTGCGCCTTATCCGGTAACTATCGTCTTGAGTCCAACCCGGTAAGACACGACTTATCGCCACTGGCAGCAGCCACTGGTAACAGGATTAGCAGAGCGAGGTATGTAGGCGGTGCTACAGAGTTCTTGAAGTGGTGGCCTAACTACGGCTACACTAGAAGGACAGTATTTGGTATCTGCGCTCTGCTGAAGCCAGTTACCTTCGGAAAAAGAGTTGGTAGCTCTTGATCCGGCAAACAAACCACCGCTGGTAGCGGTGGTTTTTTTGTTTGCAAGCAGCAGATTACGCGCAGAAAAAAAGGATCTCAAGAAGATCCTTTGATCTTTTCTACGGGGTCTGACGCTCAGTGGAACGAAAACTCACGTTAAGGGATTTTGGTCATGAGATTATCAAAAAGGATCTTCACCTAGATCCTTTTAAATCAATCTAAAGTATATATGAGTAAACTTGGTCTGACAGTTACCAATGCTTAATCAGTGAGGCACCTATCTCAGCGATCTGTCTATTTCGTTCATCCATAGTTGCCTGACTCCCCGTCGTGTAGATAACTACGATACGGGAGGGCTTACCATCTGGCCCCAGTGCTGCAATGATACCGCGAGACCCACGCTCACCGGCTCCAGATTTATCAGCAATAAACCAGCCAGCCGGAAGGGCCGAGCGCAGAAGTGGTCCTGCAACTTTATCCGCCTCCATCCAGTCTATTAATTGTTGCCGGGAAGCTAGAGTAAGTAGTTCGCCAGTTAATAGTTTGCGCAACGTTGTTGCCATTGNTGCAGGCATCGTGGTGTCACGCTCGTCGTTTGGTATGGCTTCATTCAGCTCCGGTTCCCAACGATCAAGGCGAGTTACATGATCCCCCATGTTGTGCAAAAAAGCGGTTAGCTCCTTCGGTCCTCCGATCGTTGTCAGAAGTAAGTTGGCCGCAGTGTTATCACTCATGGTTATGGCAGCACTGCATAATTCTCTTACTGTCATGCCATCCGTAAGATGCTTTTCTGTGACTGGTGAGTACTCAACCAAGTCATTCTGAGAATAGTGTATGCGGCGACCGAGTTGCTCTTGCCCGGCGTCAACACGGGATAATACCGCGCCACATAGCAGAACTTTAAAAGTGCTCATCATTGGAAAACGTTCTTCGGGGCGAAAACTCTCAAGGATCTTACCGCTGTTGAGATCCAGTTCGATGTAACCCACTCGTGCACCCAACTGATCTTCAGCATCTTTTACTTTCACCAGCGTTTCTGGGTGAGCAAAAACAGGAAGGCAAAATGCCGCAAAAAAGGGAATAAGGGCGACACGGAAATGTTGAATACTCATACTCTTCCTTTTTCAATATTATTGAAGCATTTATCAGGGTTATTGTCTCATGAGCGGATACATATTTGAATGTATTTAGAAAAATAAACAAATAGGGGTTCCGCGCACATTTCCCCGAAAAGTGCCACCTGACGTCTAAGAAACCATTATTATCATGACATTAACCTATAAAAATAGGCGTATCACGAGGCCCTTTCGTCTTCAAGGATCCGAATTCCCGGGAGAGCTCGATATCGCATGCGGATTTAAATTAATTAA

[1244] TABLE 12 Nucleotide sequence of pIB/V5-His-DEST. OpIE-2 pr 1CATGATGATA AACAATGTAT GGTGCTAATG TTGCTTCAAC AACAATTCTG GTACTACTATTTGTTACATA CCACGATTAC AACGAAGTTG TTGTTAAGAC 51 TTGAACTGTG TTTTCATGTTTGCCAACAAG CACCTTTATA CTCGGTGGCC AACTTGACAC AAAAGTACAA ACGGTTGTTCGTGGAAATAT GAGCCACCGG 101 TCCCCACCAC CAACTTTTTT GCACTGCAAA AAAACACGCTTTTGCACGCG AGGGGTGGTG GTTGAAAAAA CGTGACGTTT TTTTGTGCGA AAACGTGCGC 151GGCCCATACA TAGTACAAAC TCTACGTTTC GTAGACTATT TTACATAAAT CCGGGTATGTATCATGTTTG AGATGCAAAG CATCTGATAA AATGTATTTA 201 AGTCTACACC GTTGTATACGCTCCAAATAC ACTACCACAC ATTGAACCTT TCAGATGTGG CAACATATGC GAGGTTTATGTGATGGTGTG TAACTTGGAA 251 TTTGCAGTGC AAAAAAGTAC GTGTCGGCAG TCACGTAGGCCGGCCTTATC AAACGTCACG TTTTTTCATG CACAGCCGTC AGTGCATCCG GCCGGAATAG 301GGGTCGCGTC CTGTCACGTA CGAATCACAT TATCGGACCG GACGAGTGTT CCCAGCGCAGGACAGTGCAT GCTTAGTGTA ATAGCCTGGC CTGCTCACAA 351 GTCTTATCGT GACAGGACGCCAGCTTCCTG TGTTGCTAAC CGCAGCCGGA CAGAATAGCA CTGTCCTGCG GTCGAAGGACACAACGATTG GCGTCGGCCT 401 CGCAACTCCT TATCGGAACA GGACGCGCCT CCATATCAGCCGCGCGTTAT GCGTTGAGGA ATAGCCTTGT CCTGCGCGGA GGTATAGTCG GCGCGCAATA 451CTCATGCACG TGACCGGACA CGAGGCGCCC GTCCCGCTTA TCGCGCCTAT GAGTACGTGCACTGGCCTGT GCTCCGCGGG CAGGGCGAAT AGCGCGGATA OpIE2FOR OpIE-2 pr 501AAATACAGCC CGCAACGATC TGGTAAACAC AGTTGAACAG CATCTGTTCG TTTATGTCGGGCGTTGCTAG ACCATTTGTG TCAACTTGTC GTAGACAAGC 551 AATTTAAAGC TTGATATCGAATTCCTGCAG CCCAGCGCTG GATCCTCGAT TTAAATTTCG AACTATAGCT TAAGGACGTCGGGTCGCGAC CTAGGAGCTA attR1 601 CACAAGTTTG TACAAAAAAG CTGAACGAGAAACGTAAAAT GATATAAATA GTGTTCAAAC ATGTTTTTTC GACTTGCTCT TTGCATTTTACTATATTTAT 651 TCAATATATT AAATTAGATT TTGCATAAAA AACAGACTAC ATAATACTGTAGTTATATAA TTTAATCTAA AACGTATTTT TTGTCTGATG TATTATGACA 701 AAAACACAACATATCCAGTC ACTATGGCGG CCGCATTAGG CACCCCAGGC TTTTGTGTTG TATAGGTCAGTGATACCGCC GGCGTAATCC GTGGGGTCCG 751 TTTACACTTT ATGCTTCCGG CTCGTATAATGTGTGGATTT TGAGTTAGGA AAATGTGAAA TACGAAGGCC GAGCATATTA CACACCTAAAACTCAATCCT Cmr 801 TCCGTCGAGA TTTTCAGGAG CTAAGGAAGC TAAAATGGAGAAAAAAATCA AGGCAGCTCT AAAAGTCCTC GATTCCTTCG ATTTTACCTC TTTTTTTAGT 851CTGGATATAC CACCGTTGAT ATATCCCAAT GGCATCGTAA AGAACATTTT GACCTATATGGTGGCAACTA TATAGGGTTA CCGTAGCATT TCTTGTAAAA 901 GAGGCATTTC AGTCAGTTGCTCAATGTACC TATAACCAGA CCGTTCAGCT CTCCGTAAAG TCAGTCAACG AGTTACATGGATATTGGTCT GGCAAGTCGA 951 GGATATTACG GCCTTTTTAA AGACCGTAAA GAAAAATAAGCACAAGTTTT CCTATAATGC CGGAAAAATT TCTGGCATTT CTTTTTATTC GTGTTCAAAA 1001ATCCGGCCTT TATTCACATT CTTGCCCGCC TGATGAATGC TCATCCGGAA TAGGCCGGAAATAAGTGTAA GAACGGGCGG ACTACTTACG AGTAGGCCTT 1051 TTCCGTATGG CAATGAAAGACGGTGAGCTG GTGATATGGG ATAGTGTTCA AAGGCATACC GTTACTTTCT GCCACTCGACCACTATACCC TATCACAAGT 1101 CCCTTGTTAC ACCGTTTTCC ATGAGCAAAC TGAAACGTTTTCATCGCTCT GGGAACAATG TGGCAAAAGG TACTCGTTTG ACTTTGCAAA AGTAGCGAGA 1151GGAGTGAATA CCACGACGAT TTCCGGCAGT TTCTACACAT ATATTCGCAA CCTCACTTATGGTGCTGCTA AAGGCCGTCA AAGATGTGTA TATAAGCGTT 1201 GATGTGGCGT GTTACGGTGAAAACCTGGCC TATTTCCCTA AAGGGTTTAT CTACACCGCA CAATGCCACT TTTGGACCGGATAAAGGGAT TTCCCAAATA 1251 TGAGAATATG TTTTTCGTCT CAGCCAATCC CTGGGTGAGTTTCACCAGTT ACTCTTATAC AAAAAGCAGA GTCGGTTAGG GACCCACTCA AAGTGGTCAA 1301TTGATTTAAA CGTGGCCAAT ATGGACAACT TCTTCGCCCC CGTTTTCACC AACTAAATTTGCACCGGTTA TACCTGTTGA AGAAGCGGGG GCAAAAGTGG 1351 ATGGGCAAAT ATTATACGCAAGGCGACAAG GTGCTGATGC CGCTGGCGAT TACCCGTTTA TAATATGCGT TCCGCTGTTCCACGACTACG GCGACCGCTA 1401 TCAGGTTCAT CATGCCGTTT GTGATGGCTT CCATGTCGGCAGAATGCTTA AGTCCAAGTA GTACGGCAAA CACTACCGAA GGTACAGCCG TCTTACGAAT 1451ATGAATTACA ACAGTACTGC GATGAGTGGC AGGGCGGGGC GTAAACGCGT TACTTAATGTTGTCATGACG CTACTCACCG TCCCGCCCCG CATTTGCGCA 1501 GGATCCGGCT TACTAAAAGCCAGATAACAG TATGCGTATT TGCGCGCTGA CCTAGGCCGA ATGATTTTCG GTCTATTGTCATACGCATAA ACGCGCGACT 1551 TTTTTGCGGT ATAAGAATAT ATACTGATAT GTATACCCGAAGTATGTCAA AAAAACGCCA TATTCTTATA TATGACTATA CATATGGGCT TCATACAGTT 1601AAAGAGGTAT GCTATGAAGC AGCGTATTAC AGTGACAGTT GACAGCGACA TTTCTCCATACGATACTTCG TCGCATAATG TCACTGTCAA CTGTCGCTGT 1651 GCTATCAGTT GCTCAAGGCATATATGATGT CAATATCTCC GGTCTGGTAA CGATAGTCAA CGAGTTCCGT ATATACTACAGTTATAGAGG CCAGACCATT 1701 GCACAACCAT GCAGAATGAA GCCCGTCGTC TGCGTGCCGAACGCTGGAAA CGTGTTGGTA CGTCTTACTT CGGGCAGCAG ACGCACGGCT TGCGACCTTT 1751GCGGAAAATC AGGAAGGGAT GGCTGAGGTC GCCCGGTTTA TTGAAATGAA CGCCTTTTAGTCCTTCCCTA CCGACTCCAG CGGGCCAAAT AACTTTACTT ccdB 1801 CGGCTCTTTTGCTGACGAGA ACAGGGGCTG GTGAAATGCA GTTTAAGGTT GCCGAGAAAA CGACTGCTCTTGTCCCCGAC CACTTTACGT CAAATTCCAA 1851 TACACCTATA AAAGAGAGAG CCGTTATCGTCTGTTTGTGG ATGTACAGAG ATGTGGATAT TTTCTCTCTC GGCAATAGCA GACAAACACCTACATGTCTC 1901 TGATATTATT GACACGCCCG GGCGACGGAT GGTGATCCCC CTGGCCAGTGACTATAATAA CTGTGCGGGC CCGCTGCCTA CCACTAGGGG GACCGGTCAC 1951 CACGTCTGCTGTCAGATAAA GTCTCCCGTG AACTTTACCC GGTGGTGCAT GTGCAGACGA CAGTCTATTTCAGAGGGCAC TTGAAATGGG CCACCACGTA 2001 ATCGGGGATG AAAGCTGGCG CATGATGACCACCGATATGG CCAGTGTGCC TAGCCCCTAC TTTCGACCGC GTACTACTGG TGGCTATACCGGTCACACGG 2051 GGTCTCCGTT ATCGGGGAAG AAGTGGCTGA TCTCAGCCAC CGCGAAAATGCCAGAGGCAA TAGCCCCTTC TTCACCGACT AGAGTCGGTG GCGCTTTTAC 2101 ACATCAAAAACGCCATTAAC CTGATGTTCT GGGGAATATA AATGTCAGGC TGTAGTTTTT GCGGTAATTGGACTACAAGA CCCCTTATAT TTACAGTCCG attR2 2151 TCCCTTATAC ACAGCCAGTCTGCAGGTCGA CCATAGTGAC TGGATATGTT AGGGAATATG TGTCGGTCAG ACGTCCAGCTGGTATCACTG ACCTATACAA 2201 GTGTTTTACA GTATTATGTA GTCTGTTTTT TATGCAAAATCTAATTTAAT CACAAAATGT CATAATACAT CAGACAAAAA ATACGTTTTA GATTAAATTA 2251ATATTGATAT TTATATCATT TTACGTTTCT CGTTCAGCTT TCTTGTACAA TATAACTATAAATATAGTAA AATGCAAAGA GCAAGTCGAA AGAACATGTT V5 tag 2301 AGTGGTGATCGACCCGGGTC TAGAGGGCCC GCGGTTCGAA GGTAAGCCTA TCACCACTAG CTGGGCCCAGATCTCCCGGG CGCCAAGCTT CCATTCGGAT Poly His 6 tag 2351 TCCCTAACCCTCTCCTCGGT CTCGATTCTA CGCGTACCGG TCATCATCAC AGGGATTGGG AGAGGAGCCAGAGCTAAGAT GCGCATGGCC AGTAGTAGTG OpIE-2 PolyA 2401 CATCACCATT GAGTTTATCTGACTAAATCT TAGTTTGTAT TGTCATGTTT GTAGTGGTAA CTCAAATAGA CTCATTTAGAATCAAACATA ACAGTACAAA 2451 TAATACAATA TGTTATGTTT AAATATGTTT TTAATAAATTTTATAAAATA ATTATGTTAT ACAATACAAA TTTATACAAA AATTATTTAA AATATTTTAT 2501ATTTCAACTT TTATTGTAAC AACATTGTCC ATTTACACAC TCCTTTCAAG TAAAGTTGAAAATAACATTG TTGTAACAGG TAAATGTGTG AGGAAAGTTC 2551 CGCGTGGGAT CGATGCTCACTCAAAGGCGG TAATACGGTT ATCCACAGAA GCGCACCCTA GCTACCAGTG AGTTTCCGCCATTATGCCAA TAGGTGTCTT pMB1 ori 2601 TCAGGGGATA ACGCAGGAAA GAACATGTGAGCAAAAGGCC AGCAAAAGGC AGTCCCCTAT TGCGTCCTTT CTTGTACACT CGTTTTCCGGTCGTTTTCCG 2651 CAGGAACCGT AAAAAGGCCG CGTTGCTGGC GTTTTTCCAT AGGCTCCGCCGTCCTTGGCA TTTTTCCGGC GCAACGACCG CAAAAAGGTA TCCGAGGCGG 2701 CCCCTGACGAGCATCACATA AATCGACGCT CAAGTCAGAG GTGGCGAAAC GGGGACTGCT CGTAGTGTTTTTAGCTGCGA GTTCAGTCTC CACCGCTTTG 2751 CCGACAGGAC TATPAAGATA CCAGGCGTTTCCCCCTGGAA GCTCCCTCGT GGCTGTCCTG ATATTTCTAT GGTCCGCAAA GGGGGACCTTCGAGGGAGCA 2801 GCGCTCTCCT GTTCCGACCC TGCCGCTTAC CGGATACCTG TCCGCCTTTCCGCGAGAGGA CAAGGCTGGG ACGGCGAATG GCCTATGGAC AGGCGGAAAG 2851 TCCCTTCGGGAAGCGTGGCG CTTTCTCATA GCTCACGCTG TAGGTATCTC AGGGAAGCCC TTCGCACCGCGAAAGAGTAT CGAGTGCGAC ATCCATAGAG 2901 AGTTCGGTGT AGGTCGTTCG CTCCAAGCTGGGCTGTGTGC ACGAACCCCC TCAAGCCACA TCCAGCAAGC GAGGTTCGAC CCGACACACGTGCTTGGGGG 2951 CGTTCAGCCC GACCGCTGCG CCTTATCCGG TAACTATCGT CTTGAGTCCAGCAAGTCGGG CTGGCGACGC GGAATAGGCC ATTGATAGCA GAACTCAGGT 3001 ACCCGGTAAGACACGACTTA TCGCCACTGG CAGCAGCCAC TGGTAACAGG TGGGCCATTC TGTGCTGAATAGCGGTGACC GTCGTCGGTG ACCATTGTCC 3051 ATTAGCAGAG CGAGGTATGT AGGCGGTGCTACAGAGTTCT TGAAGTGGTG TAATCGTCTC GCTCCATACA TCCGCCACGA TGTCTCAAGAACTTCACCAC 3101 GCCTAACTAC GGCTACACTA GAAGAACAGT ATTTGGTATC TGCGCTCTGCCGGATTGATG CCGATGTGAT CTTCTTGTCA TAAACCATAG ACGCGAGACG 3151 TGAAGCCAGTTACCTTCGGA AAAAGAGTTG GTAGCTCTTG ATCCGGCAAA ACTTCGGTCA ATGGAAGCCTTTTTCTCAAC CATCGAGAAC TAGGCCGTTT 3201 CAAACCACCG CTGGTAGCGG TGGTTTTTTTGTTTGCAAGC AGCAGATTAC GTTTGGTGGC GACCATCGCC ACCAAAAAAA CAAACGTTCGTCGTCTAATG 3251 GCGCAGAAAA AAAGGATCTC AAGAAGATCC TTTGATCTTT TCTACGGGGTCGCGTCTTTT TTTCCTAGAG TTCTTCTAGG AAACTAGAAA AGATGCCCCA 3301 CTGACGCTCAGTGGAACGAA AACTCACGTT AAGGGATTTT GGTCATGCCC GACTGCGAGT CACCTTGCTTTTGAGTGCAA TTCCCTAAAA CCAGTACGGG GP64 promoter 3351 TTGTTCCGAAGGGTTGTGTC ACGTAGGCCA GATAACGGTC GGGTATATAA AACAAGGCTT CCCAACACAGTGCATCCGGT CTATTGCCAG CCCATATATT 3401 GATGCCTCAA TGCTACTAGT AAATCAGTCACACCAAGGCT TCAATAAGGA CTACGGAGTT ACGATGATCA TTTAGTCAGT GTGGTTCCGAAGTTATTCCT EM7 3451 ACACACAAGC AAGCCCTTTG AGTCAAGGGC TGCCGGGCTGCAGCACGTGT TGTGTGTTCG TTCGGGAAAC TCAGTTCCCG ACGGCCCGAC GTCGTGCACA 3501TGACAATTAA TCATCGGCAT AGTATATCGG CATAGTATAA TACGACPAGG ACTGTTAATTAGTAGCCGTA TCATATAGCC GTATCATATT ATGCTGTTCC Blasticidin (r) 3551TGAGGAACTA AACCATGGCC AAGCCTTTGT CTCAAGAAGA ATCCACCCTC ACTCCTTGATTTGGTACCGG TTCGGAAACA GAGTTCTTCT TAGGTGGGAG 3601 ATTGAAAGAG CAACGGCTACAATCAACAGC ATCCCCATCT CTGAAGACTA TAACTTTCTC GTTGCCGATG TTAGTTGTCGTAGGGGTAGA GACTTCTGAT 3651 CAGCGTCGCC GGCGCAGCTC TCTCTAGCGA CGGCCGCATCTTCACTGGTG GTCGCAGCGG CCGCGTCGAG AGAGATCGCT GCCGGCGTAG AAGTGACCAC 3701TCAATGTATA TCATTTTACT GGGGGACCTT GCGCAGAACT CGTGGTGCTG AGTTACATATAGTAAAATGA CCCCCTGGAA CGCGTCTTGA GCACCACGAC 3751 GGCACTGCTG CTGCTGCGGCAGCTGGCAAC CTGACTTGTA TCGTCGCGAT CCGTGACGAC GACGACGCCG TCGACCGTTGGACTGAACAT AGCAGCGCTA 3801 CGGAAATGAG AACAGGGGCA TCTTGAGCCC CTGCGGACGGTGCCGACAGG GCCTTTACTC TTGTCCCCGT AGAACTCGGG GACGCCTGCC ACGGCTGTCC 3851TTCTTCTCGA TCTGCATCCT GGGATCAAAG CCATAGTGAA GGACAGTGAT AAGAAGAGCTAGACGTAGGA CCCTAGTTTC GGTATCACTT CCTGTCACTA 3901 GGACAGCCGA CGGCAGTTGGGATTCGTGAA TTGCTGCCCT CTGGTTATGT CCTGTCGGCT GCCGTCAACC CTAAGCACTTAACGACGGGA GACCAATACA 3951 GTGGGAGGGC TAAGCACTTC GTGGCCGAGG AGCAGGACTGACACGTCCCG CACCCTCCCG ATTCGTGAAG CACCGGCTCC TCGTCCTGAC TGTGCAGGGC 4001GGAGATCTGC ATGTCTACTA AACTCACAAA TTAGAGCTTC AATTTAATTA CCTCTAGACGTACAGATGAT TTGAGTGTTT AATCTCGAAG TTAAATTAAT Amp (r) 4051 TATCAGTTATTACCCATTGA AAAAGGAAGA GTATGAGTAT TCAACATTTC ATAGTCAATA ATGGGTAACTTTTTCCTTCT CATACTCATA AGTTGTAAAG 4101 CGTGTCGCCC TTATTCCCTT TTTTGCGGCATTTTGCCTTC CTGTTTTTGC GCACAGCGGG AATAAGGGAA AAAACGCCGT AAAACGGAAGGACAAAAACG 4151 TCACCCAGAA ACGCTGGTGA AAGTAAAAGA TGCTGAAGAT CACTTGGGTGAGTGGGTCTT TGCGACCACT TTCATTTTCT ACGACTTCTA GTCAACCCAC 4201 CACGAGTGGGTTACATCGAA CTGGATCTCA ACAGCGGTAA GATCCTTGAG GTGCTCACCC AATGTAGCTTGACCTAGAGT TGTCGCCATT CTAGGAACTC 4251 AGTTTTCGCC CCGAAGAACG TTTTCCAATGATGAGCACTT TTAAAGTTCT TCAAAAGCGG GGCTTCTTGC AAAAGGTTAC TACTCGTGAAAATTTCAAGA 4301 GCTATGTGGC GCGGTATTAT CCCGTATTGA CGCCGGGCAA GAGCAACTCGCGATACACCG CGCCATAATA GGGCATAACT GCGGCCCGTT CTCGTTGAGC 4351 GTCGCCGCATACACTATTCT CAGAATGACT TGGTTGAGTA CTCACCAGTC CAGCGGCGTA TGTGATAAGAGTCTTACTGA ACCAACTCAT GAGTGGTCAG 4401 ACAGAAAAGC ATCTTACGGA TGGCATGACAGTAAGAGAAT TATGCAGTGC TGTCTTTTCG TAGAATGCCT ACCGTACTGT CATTCTCTTAATACGTCACG 4451 TGCCATAACC ATGAGTGATA ACACTGCGGC CAACTTACTT CTGACAACGAACGGTATTGG TACTCACTAT TGTGACGCCG GTTGAATGAA GACTGTTGCT 4501 TCGGAGGACCGAAGGAGCTA ACCGCTTTTT TGCACAACAT GGGGGATCAT AGCCTCCTGG CTTCCTCGATTGGCGAAAAA ACGTGTTGTA CCCCCTAGTA 4551 GTAACTCGCC TTGATCGTTG GGAACCGGAGCTGAATGAAG CCATACCAAA CATTGAGCGG AACTAGCAAC CCTTGGCCTC GACTTACTTCGGTATGGTTT 4601 CGACGAGCGT GACACCACGA TGCCTGTAGC AATGGCAACA ACGTTCCGCAGCTGCTCGCA CTGTGGTGCT ACGGACATCG TTACCGTTGT TGCAACGCGT 4651 AACTATTAACTGGCGAACTA CTTACTCTAG CTTCCCGGCA ACAATTAATA TTGATAATTG ACCGCTTGATGAATGAGATC GAAGGGCCGT TGTTAATTAT 4701 GACTGGATGG AGGCGGATAA AGTTGCAGGACCACTTCTGC GCTCGGCCCT CTGACCTACC TCCGCCTATT TCAACGTCCT GGTGAAGACGCGAGCCGGGA 4751 TCCGGCTGGC TGGTTTATTG CTGATAAATC TGGAGCCGGT GAGCGTGGGTAGGCCGACCG ACCAAATAAC GACTATTTAG ACCTCGGCCA CTCGCACCCA 4801 CTCGCGGTATCATTGCAGCA CTGGGGCCAG ATGGTAAGCC CTCCCGTATC GAGCGCCATA GTAACGTCGTGACCCCGGTC TACCATTCGG GAGGGCATAG 4851 GTAGTTATCT ACACGACGGG GAGTCAGGCAACTATGGATG AACGAAATAG CATCAATAGA TGTGCTGCCC CTCAGTCCGT TGATACCTACTTGCTTTATC 4901 ACAGATCGCT GAGATAGGTG CCTCACTGAT TAAGCATTGG TAACTGTCAGTGTCTAGCGA CTCTATCCAC GGAGTGACTA ATTCGTAACC ATTGACAGTC 4951 ACCAAGTTTACTCATATATA CTTTAGATTG ATTTAAAACT TCATTTTTAA TGGTTCAAAT GAGTATATATGAAATCTAAC TAAATTTTGA AGTAAAAATT 5001 TTTAAAAGGA TCTAGGTGAA GATCCTTTTTGATAATCT AAATTTTCCT AGATCCACTT CTAGGAAAAA CTATTAGA

[1245] TABLE 13 Nucleotide sequence of the V5-His DEST cassette. phpromoter ----- 1 ATAAGTATTT TACTGTTTTC GTAACAGTTT TGTAATAAAA AAACCTATAATATTCATAAA ATGACAAAAG CATTGTCAAA ACATTATTTT TTTGGATATT 51 ATATTCCGGATTATTCATAC CGTCCCACCA TCGGGCGCGG ATCCCCGGGT TATAAGGCCT AATAAGTATGGCAGGGTGGT AGCCCGCGCC TAGGGGCCCA     att R1         --------------------------------------------- 101 ACCGATATCACAAGTTTGTA CAAAAAAGCT GAACGAGAAA CGTAAAATGA TGGCTATAGT GTTCAAACATGTTTTTTCGA CTTGCTCTTT GCATTTTACT att R1------------------------------------------------------ 151 TATAAATATCAATATATTAA ATTAGATTTT GCATAAAAAA CAGACTACAT ATATTTATAG TTATATAATTTAATCTAAAA CGTATTTTTT GTCTGATGTA att R1 ------- 201 AATACTGTAAAACACAACAT ATCCAGTCAC TATGGCGGCC GCTCCCTAAC TTATGACATT TTGTGTTGTATAGGTCAGTG ATACCGCCGG CGAGGGATTG 251 CCACGGGGCC CGTGGCTATG GCAGGGCTTGCCGCCCCGAC GTTGGCTGCG GGTGCCCCGG GCACCGATAC CGTCCCGAAC GGCGGGGCTGCAACCGACGC 301 AGCCCTGGGC CTTCACCCGA ACTTGGGGGT TGGGGTGGGG AAAAGGAAGATCGGGACCCG GAAGTGGGCT TGAACCCCCA ACCCCACCCC TTTTCCTTCT 351 AACGCGGGCGTATTGGTCCC AATGGGGTCT CGGTGGGGTA TCGACAGAGT TTGCGCCCGC ATAACCAGGGTTACCCCAGA GCCACCCCAT AGCTGTCTCA 401 GCCAGCCCTG GGACCGAACC CCGCGTTTATGAACAAACGA CCCAACACCC CGGTCGGGAC CCTGGCTTGG GGCGCAAATA CTTGTTTGCTGGGTTGTGGG 451 GTGCGTTTTA TTCTGTCTTT TTATTGCCGT CATAGCGCGG GTTCCTTCCGCACGCAAAAT AAGACAGAAA AATAACGGCA GTATCGCGCC CAAGGAAGGC 501 GTATTGTCTCCTTCCGTGTT TCAGTTAGCC TCCCCCATCT CCCGGGCAAA CATAACAGAG GAAGGCACAAAGTCAATCGG AGGGGGTAGA GGGCCCGTTT    ---------------------------   tkgene     N  A  E   G  M  E   R    A  F 551 CGTGCGCGCC AGGTCGCAGATCGTCGGTAT GGAGCCTGGG GTGGTGACGT GCACGCGCGG TCCAGCGTCT AGCAGCCATACCTCGGACCC CACCACTGCA------------------------------------------------------           tk gene T  R  A  L   D  C  I   T  P  I   S  G  P  T   T  V  H . 601 GGGTCTGGACCATCCCGGAG GTAAGTTGCA GCAGGGCGTC CCGGCAGCCG CCCAGACCTG GTAGGGCCTCCATTCAACGT CGTCCCGCAG GGCCGTCGGC------------------------------------------------------           tk gene. T  Q  V   M  G  S  T   L  Q  L   L  A  D   R  C  G  A . 651 GCGGGCGATTGGTCGTAATC CAGGATAAAG ACATGCATGG GACGGAGGCG CGCCCGCTAA CCAGCATTAGGTCCTATTTC TGTACGTACC CTGCCTCCGC------------------------------------------------------           tk gene..  P  S  Q   D  Y  D   L  I  F  V   H  M  P   R  L  R 701 TTTGGCCAAGACGTCCAAAG CCCAGGCAAA CACGTTATAC AGGTCGCCGT AAACCGGTTC TGCAGGTTTCGGGTCCGTTT GTGCAATATG TCCAGCGGCA------------------------------------------------------           tk geneK  A  L  V   D  L  A   W  A  F   V  N  Y  L   D  G  N . 751 TGGGGGCCAGCAACTCGGGG GCCCGAAACA GGGTAAATAA CGTGTCCCCG ACCCCCGGTC GTTGAGCCCCCGGGCTTTGT CCCATTTATT GCACAGGGGC------------------------------------------------------           tk gene. P  A  L   L  E  P  A   R  F  L   T  F  L   T  D  G  I . 801 ATATGGGGTCGTGGGCCCGC GTTGCTCTGG GGCTCGGCAC CCTGGGGCGG TATACCCCAG CACCCGGGCGCAACGAGACC CCGAGCCGTG GGACCCCGCC------------------------------------------------------           tk gene.. H  P  R   P  G  A   N  S  Q  P   E  A  G   Q  P  P 851 CACGGCCGCCCCCGAAAGCT GTCCCCAATC CTCCCGCCAC GACCCGCCGC GTGCCGGCGG GGGCTTTCGACAGGGGTTAG GAGGGCGGTG CTGGGCGGCG------------------------------------------------------           tk gene V  A  A  G   S  L  Q   G  W  D   E  R  W  S   G  G  G . 901 CCTGCAGATACCGCACCGTA TTGGCAAGCA GCCCATAAAC GCGGCGAATC GGACGTCTAT GGCGTGGCATAACCGTTCGT CGGGTATTTG CGCCGCTTAG------------------------------------------------------           tk gene. Q  L  Y   R  V  T  N   A  L  L   G  Y  V   R  R  I  A . 951 GCGGCCAGCATAGCCAGGTC AAGCCGCTCG CCGGGGCGCT GGCGTTTGGC CGCCGGTCGT ATCGGTCCAGTTCGGCGAGC GGCCCCGCGA CCGCAAACCG------------------------------------------------------           tk gene.. A  L  M   A  L  D   L  R  E  G   P  R  Q   R  K  A 1001 CAGGCGGTCGATGTGTCTGT CCTCCGGAAG GGCCCCCAAC ACGATGTTTG GTCCGCCAGC TACACAGACAGGAGGCCTTC CCGGGGGTTG TGCTACAAAC------------------------------------------------------           tk gene L  R  D  I   H  R  D   E  P  L   A  G  L  V   I  N  T . 1051 TGCCGGGCAAGGTCGGCGGG ATGAGGGCCA CGAACGCCAG CACGGCCTGG ACGGCCCGTT CCAGCCGCCCTACTCCCGGT GCTTGCGGTC GTGCCGGACC------------------------------------------------------           tk gene. G  P  L   T  P  P  I   L  A  V   F  A  L   V  A  Q  P . 1101GGGGTCATGC TGCCCATAAG GTATCGCGCG GCCGGGTAGC ACAGGAGGGC CCCCAGTACGACGGGTATTC CATAGCGCGC CGGCCCATCG TGTCCTCCCG------------------------------------------------------           tk gene.. T  M  S   G  M  L   Y  R  A  A   P  Y  C   L  L  A 1151 GGCGATGGGATGGCGGTCGA AGATGAGGGT GAGGGCCGGG GGCGGGGCAT CCGCTACCCT ACCGCCAGCTTCTACTCCCA CTCCCGGCCC CCGCCCCGTA------------------------------------------------------           tk gene A  I  P  H   R  D  F   I  L  T   L  A  P  P   P  A  H . 1201 GTGAGCTCCCAGCCTCCCCC CCGATATGAG GAGCCAGAAC GGCGTCGGTC CACTCGAGGG TCGGAGGGGGGGCTATACTC CTCGGTCTTG CCGCAGCCAG------------------------------------------------------           tk gene. S  S  G   A  E  G  G   I  H  P   A  L  V   A  D  T  V . 1251ACGGCATAAG GCATGCCCAT TGTTATCTGG GCGCTTGTCA TTACCACCGC TGCCGTATTCCGTACGGGTA ACAATAGACC CGCGAACAGT AATGGTGGCG------------------------------------------------------           tk gene.. A  Y  P   M  G  M   T  I  Q  A   S  T  M   V  V  A 1301 CGCGTCCCCGGCCGATATCT CACCCTGGTC GAGGCGGTGT TGTGTGGTGT GCGCAGGGGC CGGCTATAGAGTGGGACCAG CTCCGCCACA ACACACCACA------------------------------------------------------           tk gene A  D  G  A   S  I  E   G  Q  D   L  R  H  Q   T  T  Y . 1351 AGATGTTCGCGATTGTCTCG GAAGCCCCCA ACACCCGCCA GTAAGTCATC TCTACAAGCG CTAACAGAGCCTTCGGGGGT TGTGGGCGGT CATTCAGTAG------------------------------------------------------           tk gene. I  N  A   I  T  E  S   A  G  L   V  R  W   Y  T  M  P . 1401GGCTCGGGTA CGTAGACGAT ATCGTCGCGC GAACCCAGGG CCACCAGCAG CCGAGCCCATGCATCTGCTA TAGCAGCGCG CTTGGGTCCC GGTGGTCGTC------------------------------------------------------           tk gene.. E  P  V   Y  V  I   D  D  R  S   G    L   A   V  L  L 1451 TTGCGTGGTGGTGGTTTTCC CCATCCCGTG GGGACCGTCT ATATAAACCC AACGCACCAC CACCAAAAGGGGTAGGGCAC CCCTGGCAGA TATATTTGGG------------------------------------------------------           tk gene Q  T  T  T   T  K  G   M  G  H   P  G  D  I   Y  V  R . 1501 GCAGTAGCGTGGGCATTTTC TGCTCCAGGC GGACTTCCGT GGCTTTTTGT CGTCATCGCA CCCGTAAAAGACGAGGTCCG CCTGAAGGCA CCGAAAAACA------------------------------------------------------           tk gene. L  L  T   P  M  K  Q   E  L  R   V  E  T   A  K  Q  Q . 1551TGCCGGCGAG GGCGCAACGC CGTACGTCGG TTGTTATGGC CGCGAGAACG ACGGCCGCTCCCGCGTTGCG GCATGCAGCC AACAATACCG GCGCTCTTGC------------------------------------------------------           tk gene.. R  R  P   R  L  A   T  R  R  N   N  H  G   R  S  R 1601 CGCAGCCTGGTCGAACGCAG ACGCGTGTTG ATGGCAGGGG TACGAAGCCA GCGTCGGACC AGCTTGCGTCTGCGCACAAC TACCGTCCCC ATGCTTCGGT------------------------------------------------------           tk gene A  A  Q  D   F  A  S   A  H  Q   H  C  P  Y   S  A  M . 1651 TAGATCCCGTTATCAATTAC TTATACTATC CGGCGCGCAA GCGAGCGTGT ATCTAGGGCA ATAGTTAATGAATATGATAG GCCGCGCGTT CGCTCGCACA          --------------------   ie-0promoter 1701 GCGCCGGAGC ACAATTGATA CTGATTTACG AGTTGGGCAA ACGGGCTTTACGCGGCCTCG TGTTAACTAT GACTAAATGC TCAACCCGTT TGCCCGAAAT------------------------------------------------------        ie-0promoter 1751 TATAGCCTGT CCCCTCCACA GCCCTAGTGC CGTGCGCAAA GTGCCTACGTATATCGGACA GGGGAGGTGT CGGGATCACG GCACGCGTTT CACGGATGCA------------------------------------------------------        ie-0promoter 1801 GACCAGGCTC TCCTACGCAT ATACAATCTT ATCTCTATAG ATAAGGTTTCCTGGTCCGAG AGGATGCGTA TATGTTAGAA TAGAGATATC TATTCCAAAG------------------------------------------------------        ie-0promoter 1851 CATATATAAA GCCTCTCGAT GGCTGAACGT GCACAGTATC GTGTTGATTTGTATATATTT CGGAGAGCTA CCGACTTGCA CGTGTCATAG CACAACTAAA------------------------------------------------------        ie-0promoter 1901 CTGAGTGCTA ACTAACAGTT ACAATGAACC GTTTTTTTCG AGAGAATAACGACTCACGAT TGATTGTCAA TGTTACTTGG CAAAAAAAGC TCTCTTATTG------------------------------------------------------        ie-0promoter 1951 ATTTTTGACG CGCCAAGGAC CGGGGGCAAG GGTCGTGCCA AATCTTTGCCTAAAAACTGC GCGGTTCCTG GCCCCCGTTC CCAGCACGGT TTAGAAACGG------------------------------------------------------        ie-0promoter 2001 AGCGCCTGCC GCCAACTCGC CGCCGTCGCC TGTTCGTCCG CCGCCAAAATTCGCGGACGG CGGTTGAGCG GCGGCAGCGG ACAAGCAGGC GGCGGTTTTA------------------------------------------------------        ie-0promoter 2051 CTAACATCAA ACCACCTACG CGCATCTCTC CGCCTAAACA GCCTATGTGCGATTGTAGTT TGGTGGATGC GCGTAGAGAG GCGGATTTGT CGGATACACG------------------------------------------------------        ie-0promoter 2101 ACCTCTCCGG CCAAGCCGTT GGAGCACAGC AGCATTGTAA GTAAAAAACCTGGAGAGGCC GGTTCGGCAA CCTCGTGTCG TCGTAACATT CATTTTTTGG------------------------------------------------------        ie-0promoter 2151 AGTCGTCAAC AGAAAAGATG GATATTTTGT GCCGCCCGAG TTTGGGAACATCAGCAGTTG TCTTTTCTAC CTATAAAACA CGGCGGGCTC AAACCCTTGT-----------------------------------------------------        ie-0promoter 2201 AGTTTGAAGG TTTGCCCGCG TACAGCGACA AACTGGATTT CAAACAAGAGTCAAACTTCC AAACGGGCGC ATGTCGCTGT TTGACCTAAA GTTTGTTCTC----------------------------------          ie-0 promoter   p10 promoter        ------------------------ 2251 CGCGATCTAC GTACCTGCAG GCCCGGGCTCAACCCAACAC AATATATTAT GCGCTAGATG CATGGACGTC CGGGCCCGAG TTGGGTTGTGTTATATAATA         p10 promoter------------------------------------------------------ 2301 AGTTAAATAAGAATTATTAT CAAATCATTT GTATATTAAT TAAAATACTA TCAATTTATT CTTAATAATAGTTTAGTAAA CATATAATTA ATTTTATGAT        p10 promoter lacZ----------------------------        --------------         M T M I T .2351 TACTGTAAAT TACATTTTAT TTACAATTCA CTCTAGAATG ACCATGATTA ATGACATTTAATGTAAAATA AATGTTAAGT GAGATCTTAC TGGTACTAAT  lacZ------------------------------------------------------.  D  S  L   A  V  V   L  Q  R  R   D  W  E   N  P  G 2401 CGGATTCACTGGCCGTCGTT TTACAACGTC GTGACTGGGA AAACCCTGGC GCCTAAGTGA CCGGCAGCAAAATGTTGCAG CACTGACCCT TTTGGGACCG  lacZ------------------------------------------------------ V  T  Q  L   N  R  L   A  A  H   P  P  F  A   S  W  R . 2451 GTTACCCAACTTAATCGCCT TGCAGCACAT CCCCCTTTCG CCAGCTGGCG CAATGGGTTG AATTAGCGGAACGTCGTGTA GGGGGAAAGC GGTCGACCGC  lacZ------------------------------------------------------. N  S  E   E  A  R  T   D  R  P   S  Q  Q   L  R  S  L . 2501TAATAGCGAA GAGGCCCGCA CCGATCGCCC TTCCCAACAG TTGCGCAGCC ATTATCGCTTCTCCGGGCGT GGCTAGCGGG AAGGGTTGTC AACGCGTCGG  lacZ------------------------------------------------------.  N  G  E   W  R  F   A  W  F  P   A  P  E   A  V  P 2551 TGAATGGCGAATGGCGCTTT GCCTGGTTTC CGGCACCAGA AGCGGTGCCG ACTTACCGCT TACCGCGAAACGGACCAAAG GCCGTGGTCT TCGCCACGGC  lacZ------------------------------------------------------     Bsu36I    --------  E   S   W   L   E  C  D   L  P  E   A  D  T  V   V  V  P . 2601 GAAAGCTGGC TGGAGTGCGATCTTCCTGAG GCCGATACTG TCGTCGTCCC CTTTCGACCG ACCTCACGCT AGAAGGACTCCGGCTATGAC AGCAGCAGGG  lacZ------------------------------------------------------. S  N  W   Q  M  H  G   Y  D  A   P  I  Y   T  N  V   T . 2651CTCAAACTGG CAGATGCACG GTTACGATGC GCCCATCTAC ACCAACGTAA GAGTTTGACCGTCTACGTGC CAATGCTACG CGGGTAGATG TGGTTGCATT  lacZ------------------------------------------------------.  Y  P  I   T  V  N   P  P  F  V   P  T  E   N  P  T 2701 CCTATCCCATTACGGTCAAT CCGCCGTTTG TTCCCACGGA GAATCCGACG GGATAGGGTA ATGCCAGTTAGGCGGCAAAC AAGGGTGCCT CTTAGGCTGC  lacZ------------------------------------------------------ G  C  Y  S   L  T  F   N V  D   E  S  W  L   Q  E  G . 2751 GGTTGTTACTCGCTCACATT TAATGTTGAT GAAAGCTGGC TACAGGAAGG CCAACAATGA GCGAGTGTAAATTACAACTA CTTTCGACCG ATGTCCTTCC  lacZ------------------------------------------------------. Q  T  R   I  I  F  D   G  V  N   S  A  F   H  L  W  C . 2801CCAGACGCGA ATTATTTTTG ATGGCGTTAA CTCGGCGTTT CATCTGTGGT GGTCTGCGCTTAATAAAAAC TACCGCAATT GAGCCGCAAA GTAGACACCA  lacZ------------------------------------------------------.  N  G  R   W  V  G   Y  G  Q  D   S  R  L   P  S  E 2851 GCAACGGGCGCTGGGTCGGT TACGGCCAGG ACAGTCGTTT GCCGTCTGAA CGTTGCCCGC GACCCAGCCAATGCCGGTCC TGTCAGCAAA CGGCAGACTT  lacZ------------------------------------------------------ F  D  L  S   A  F  L   R  A  G   E  N  R  L   A  V  M . 2901 TTTGACCTGAGCGCATTTTT ACGCGCCGGA GAAAACCGCC TCGCGGTGAT AAACTGGACT CGCGTAAAAATGCGCGGCCT CTTTTGGCGG AGCGCCACTA  lacZ------------------------------------------------------. V  L  R   W  S  D  G   S  Y  L   E  D  Q   D  M  W  R . 2951GGTGCTGCGT TGGAGTGACG GCAGTTATCT GGAAGATCAG GATATGTGGC CCACGACGCAACCTCACTGC CGTCAATAGA CCTTCTAGTC CTATACACCG  lacZ------------------------------------------------------.  M  S  G    I  F  R   D  V  S  L   L  H  K   P  T  T 3001 GGATGAGCGGCATTTTCCGT GACGTCTCGT TGCTGCATAA ACCGACTACA CCTACTCGCC GTAAAAGGCACTGCAGAGCA ACGACGTATT TGGCTGATGT  lacZ------------------------------------------------------ Q  I  S  D   F  H  V   A  T  R   F  N  D  D   F  S  R . 3051 CAAATCAGCGATTTCCATGT TGCCACTCGC TTTAATGATG ATTTCAGCCG GTTTAGTCGC TAAAGGTACAACGGTGAGCG AAATTACTAC TAAAGTCGGC  lacZ------------------------------------------------------. A  V  L   E  A  E  V   Q  M  C   G  E  L   R  D  Y  L . 3101CGCTGTACTG GAGGCTGAAG TTCAGATGTG CGGCGAGTTG CGTGACTACC GCGACATGACCTCCGACTTC AAGTCTACAC GCCGCTCAAC GCACTGATGG  lacZ------------------------------------------------------.  R  V  T   V  S  L   W  Q  G  E   T  Q  V   A  S  G 3151 TACGGGTAACAGTTTCTTTA TGGCAGGGTG AAACGCAGGT CGCCAGCGGC ATGCCCATTG TCAAAGAAATACCGTCCCAC TTTGCGTCCA GCGGTCGCCG  lacZ------------------------------------------------------ T  A  P  F   G  G  E   I  I  D   E  R  G  G   Y  A  D . 3201 ACCGCGCCTTTCGGCGGTGA AATTATCGAT GAGCGTGGTG GTTATGCCGA TGGCGCGGAA AGCCGCCACTTTAATAGCTA CTCGCACCAC CAATACGGCT  lacZ------------------------------------------------------. R  V  T   L  R  L  N   V  E  N   P  K  L   W  S  A  E . 3251TCGCGTCACA CTACGTCTGA ACGTCGAAAA CCCGAAACTG TGGAGCGCCG AGCGCAGTGTGATGCAGACT TGCAGCTTTT GGGCTTTGAC ACCTCGCGGC  lacZ------------------------------------------------------.  I  P  N   L  Y  R   A  V  V  E   L  H  T   A  D  G 3301 AAATCCCGAATCTCTATCGT GCGGTGGTTG AACTGCACAC CGCCGACGGC TTTAGGGCTT AGAGATAGCACGCCACCAAC TTGACGTGTG GCGGCTGCCG  lacZ------------------------------------------------------ T  L  I  E   A  E  A   C  D  V   G  F  R  E   V  R  I . 3351 ACGCTGATTGAAGCAGAAGC CTGCGATGTC GGTTTCCGCG AGGTGCGGAT TGCGACTAAC TTCGTCTTCGGACGCTACAG CCAAAGGCGC TCCACGCCTA  lacZ------------------------------------------------------. E  N  G   L  L  L  L   N  G  K   P  L  L   I  R  G  V . 3401TGAAAATGGT CTGCTGCTGC TGAACGGCAA GCCGTTGCTG ATTCGAGGCG ACTTTTACCAGACGACGACG ACTTGCCGTT CGGCAACGAC TAAGCTCCGC  lacZ------------------------------------------------------.  N  R  H   E  H  H   P  L  H  G   Q  V  M   D  L  Q 3451 TTAACCGTCACGAGCATCAT CCTCTGCATG GTCAGGTCAT GGATGAGCAG AATTGGCAGT GCTCGTAGTAGGAGACGTAC CAGTCCAGTA CCTACTCGTC        lacZ------------------------------------------------------ T  M  V  Q   D  I  L   L  M  K   Q  N  N  F   N  A  V . 3501 ACGATGGTGCAGGATATCCT GCTGATGAAG CAGAACAACT TTAACGCCGT TGCTACCACG TCCTATAGGACGACTACTTC GTCTTGTTGA AATTGCGGCA  lacZ------------------------------------------------------. R  C  S   H  Y  P  N   H  P  L   W  Y  T   L  C  D  R . 3551GCGCTGTTCG CATTATCCGA ACCATCCGCT GTGGTACACG CTGTGCGACC CGCGACAAGCGTAATAGGCT TGGTAGGCGA CACCATGTGC GACACGCTGG  lacZ------------------------------------------------------.  Y  G  L   Y  V  V   D  E  A  N   I  E  T   H  G  M 3601 GCTACGGCCTGTATGTGGTG GATGAAGCCA ATATTGAAAC CCACGGCATG CGATGCCCGA CATACACCACCTACTTCGGT TATAACTTTG GGTGCCGTAC  lacZ------------------------------------------------------ V  P  M  N   R  L  T   D  D  P   R  W  L  P   A  M  S . 3651 GTGCCAATGAATCGTCTGAC CGATGATCCG CGCTGGCTAC CGGCGATGAG CACGGTTACT TAGCAGACTGGCTACTAGGC GCGACCGATG GCCGCTACTC  lacZ------------------------------------------------------. E  R  V   T  R  M  V   Q  R  D   R  N  H   P  S  V  I . 3701CGAACGCGTA ACGCGAATGG TGCAGCGCGA TCGTAATCAC CCGAGTGTGA GCTTGCGCATTGCGCTTACC ACGTCGCGCT AGCATTAGTG GGCTCACACT  lacZ------------------------------------------------------.  I  W  S   L  G  N   E  S  G  H   G  A  N   H  D  A 3751 TCATCTGGTCGCTGGGGAAT GAATCAGGCC ACGGCGCTAA TCACGACGCG AGTAGACCAG CGACCCCTTACTTAGTCCGG TGCCGCGATT AGTGCTGCGC  lacZ------------------------------------------------------ L  Y  R  W   I  K  S   V  D  P   S  R  P  V   Q  Y  E . 3801 CTGTATCGCTGGATCAAATC TGTCGATCCT TCCCGCCCGG TGCAGTATGA GACATAGCGA CCTAGTTTAGACAGCTAGGA AGGGCGGGCC ACGTCATACT  lacZ------------------------------------------------------. G  G  G   A  D  T  T   A  T  D   I  I  C   P  M  Y  A . 3851AGGCGGCGGA GCCGACACCA CGGCCACCGA TATTATTTGC CCGATGTACG TCCGCCGCCTCGGCTGTGGT GCCGGTGGCT ATAATAAACG GGCTACATGC  lacZ------------------------------------------------------.  R  V  D   E  D  Q   P  F  P  A   V  P  K   W  S  I 3901 CGCGCGTGGATGAAGACCAG CCCTTCCCGG CTGTGCCGAA ATGGTCCATC GCGCGCACCT ACTTCTGGTCGGGAAGGGCC GACACGGCTT TACCAGGTAG  lacZ------------------------------------------------------ K  K  W  L   S  L  P   G  E  T   R  P  L  I   L  C  E . 3951 AAAAAATGGCTTTCGCTACC TGCAGAGACG CCCCCGCTGA TCCTTTGCCA TTTTTTACCG AAAGCGATGGACCTCTCTGC GCGGGCCACT AGGAAACGCT  lacZ------------------------------------------------------. Y  A  H   A  M  G  N   S  L  G   G  F  A   K  Y  W  Q . 4001ATACGCCCAC GCGATGCGTA ACAGTCTTGG CGGTTTCGCT AAATACTGGC TATGCGGGTGCGCTACCCAT TGTCAGAACC GCCAAAGCGA TTTATGACCG  lacZ------------------------------------------------------.  A  F  R   Q  Y  P   R  L  Q  G   G  F  V   W  D  W 4051 AGGCGTTTCGTCAGTATCCC CGTTTACAGG GCGGCTTCGT CTGGGACTCG TCCGCAAAGC AGTCATACGGGCAAATGTCC CGCCGAACCA CACCCTGACC  lacZ------------------------------------------------------ V  D  Q  S   L  I  K   Y  D  E   N  G  N  P   W  S  A . 4101 GTGCATCAGTCGCTGATTAA ATATGATCAA AACGCCAACC CGTGGTCGGC CACCTAGTCA GCCACTAATTTATACTACTT TTGCCCTTGC GCACCAGCCC  lacZ------------------------------------------------------. Y  G  G   D  F  G  D   T  P  N   D  R  Q   F  C  M  N . 4151TTACCCCCCT CATTTTCCCG ATACCCCCAA CCATCCCCAC TTCTCTATCA AATCCCCCCACTAAAACCCC TATCCCCCTT CCTAGCCCTC AACACATACT  lacZ------------------------------------------------------.  G  L  V   F  A  D   R  T  P  H   P  A  L   T  E  A 4201 ACCCTCTCCTCTTTGCCCAC CCCACCCCCC ATCCACCCCT CACCCAAGCA TGCCAGACCA CAAACCCCTGCCCTCCCCCC TACCTCGCCA CTCCCTTCCT  lacZ------------------------------------------------------ K  H  Q  Q   Q  F  F   Q  F  R   L  S  G  Q   T  I  E . 4251 AAACACCACCACCAGTTTTT CCACTTCCCT TTATCCGCCC AAACCATCCA TTTGTCCTCG TCGTCAAAAACCTCAACCCA AATACCCCCC TTTCCTACCT  lacZ------------------------------------------------------. V  T  S   E  Y  L  F   R  H  S   D  N  E   L  L  H  W . 4301ACTCACCACC CAATACCTCT TCCCTCATAC CGATAACCAC CTCCTCCACT TCACTCCTCGCTTATCCACA AGCCACTATC CCTATTGCTC CACCACCTCA  lacZ------------------------------------------------------.  M  V  A   L  D  G   K  P  L  A   S  G  E   V  P  L 4351 GGATCCTCCCCCTCCATCCT AACCCCCTCC CAAGCCCTCA ACTCCCTCTG CCTACCACCC CCACCTACCATTCCCCCACC CTTCCCCACT TCACCCACAC  lacZ------------------------------------------------------ D  V  A  P   Q  G  K   Q  L  I   E  L  P  E   L  P  Q . 4401 GATGTCGCTCCACAAGGTAA ACAGTTGATT GAACTGCCTG AACTACCGCA CTACAGCGAG GTGTTCCATTTGTCAACTAA CTTGACGGAC TTGATGGCGT  lacZ------------------------------------------------------. P  E  S   A  G  Q  L   W  L  T   V  R  V   V  Q  P  N . 4451GCCGGAGAGC GCCGGGCAAC TCTGGCTCAC AGTACGCGTA GTGCAACCGA CGGCCTCTCGCGGCCCGTTG AGACCGAGTG TCATGCGCAT CACGTTGGCT  lacZ------------------------------------------------------.  A  T  A   W  S  E   A  G  H  I   S  A  W   Q  Q  W 4501 ACGCGACCGCATGGTCAGAA GCCGGGCACA TCAGCGCCTG GCAGCAGTGG TGCGCTGGCG TACCAGTCTTCGGCCCGTGT AGTCGCGGAC CGTCGTCACC  lacZ------------------------------------------------------ R  L  A  E   N  L  S   V  T  L   P  A  A  S   H  A  I . 4551 CGTCTGGCGGAAAACCTCAG TGTGACGCTC CCCGCCGCGT CCCACGCCAT GCAGACCGCC TTTTGGAGTCACACTGCGAG GGGCGGCGCA GGGTGCGGTA  lacZ------------------------------------------------------. P  H  L   T  T  S  E   M  D  F   C  I  E   L  G  N  K . 4601CCCGCATCTG ACCACCAGCG AAATGGATTT TTGCATCGAG CTGGGTAATA GGGCGTAGACTGGTGGTCGC TTTACCTAAA AACGTAGCTC GACCCATTAT  lacZ------------------------------------------------------.  R  W  Q   F  N  R   Q  S  G  F   L  S  Q   M  W  I 4651 AGCGTTGGCAATTTAACCGC CAGTCAGGCT TTCTTTCACA GATGTGGATT TCGCAACCGT TAAATTGGCGGTCAGTCCGA AAGAAAGTGT CTACACCTAA  lacZ------------------------------------------------------ G  D  K  K   Q  L  L   T  P  L   R  D  Q  F   T  R  A . 4701 GGCGATAAAAAACAACTGCT GACGCCGCTG CGCGATCAGT TCACCCGTGC CCGCTATTTT TTGTTGACGACTGCGGCGAC GCGCTAGTCA AGTGGGCACG  lacZ------------------------------------------------------. P  L  D   N  D  I  G   V  S  E   A  T  R   I  D  P  N . 4751ACCGCTGGAT AACGACATTG GCGTAAGTGA AGCGACCCGC ATTGACCCTA TGGCGACCTATTGCTGTAAC CGCATTCACT TCGCTGGGCG TAACTGGGAT  lacZ------------------------------------------------------.  A  W  V   E  R  W   K  A  A  G   H  Y  Q   A  E  A 4801 ACGCCTGGGTCGAACGCTGG AAGGCGGCGG GCCATTACCA GGCCGAAGCA TGCGGACCCA GCTTGCGACCTTCCGCCGCC CGGTAATGGT CCGGCTTCGT  lacZ------------------------------------------------------ A  L  L  Q   C  T  A   D  T  L   A  D  A  V   L  I  T . 4851 GCGTTGTTGCAGTGCACGGC AGATACACTT GCTGATGCGG TGCTGATTAC CGCAACAACG TCACGTGCCGTCTATGTGAA CGACTACGCC ACGACTAATG  lacZ------------------------------------------------------. T  A  H   A  W  Q  H   Q  G  K   T  L  F   I  S  R  K . 4901GACCGCTCAC GCGTGGCAGC ATCAGGGGAA AACCTTATTT ATCAGCCGGA CTGGCGAGTGCGCACCGTCG TAGTCCCCTT TTGGAATAAA TAGTCGGCCT  lacZ------------------------------------------------------.  T  Y  R   I  D  G   S  G  Q  M   A  I  T   V  D  V 4951 AAACCTACCGGATTGATGGT AGTGGTCAAA TGGCGATTAC CGTTGATGTT TTTGGATGGC CTAACTACCATCACCAGTTT ACCGCTAATG GCAACTACAA  lacZ------------------------------------------------------ E  V  A  S   D  T  P   H  P  A   R  I  G  L   N  C  Q . 5001 GAAGTGGCGAGCGATACACC GCATCCGGCG CGGATTGGCC TGAACTGCCA CTTCACCGCT CGCTATGTGGCGTAGGCCGC GCCTAACCGG ACTTGACGGT  lacZ------------------------------------------------------. L  A  Q   V  A  E  R   V  N  W   L  G  L   G  P  Q  E . 5051GCTGGCGCAG GTACCAGAGC GGGTAAACTC GCTCGGATTA GGGCCGCAAG CGACCGCGTCCATCGTCTCG CCCATTTGAC CGACCCTAAT CCCCGCGTTC  lacZ------------------------------------------------------.  N  Y  P   D  R  L   T  A  A  C   F  D  R   W  D  L 5101 AAAACTATCCCGACCGCCTT ACTCCCGCCT GTTTTCACCG CTGGGATCTG TTTTGATAGG CCTGGCGGAATGACGGCCGA CAAAACTGGC GACCCTAGAC  lacZ------------------------------------------------------ P  L  S  D   M  Y  T   P  Y  V   F  P  S  E   N  G  L . 5151 CCATTCTCAGACATGTATAC CCCGTACGTC TTCCCGAGCC AAAACGGTCT GGTAACAGTC TCTACATATCGCGCATGCAC AAGGCCTCCC TTTTGCCAGA  lacZ------------------------------------------------------. R  C  G   T  R  E  L   N  Y  G   P  H  Q   W  R  G  D . 5201GCGCTGCCGG ACGCGCGAAT TGAATTATGG CCCACACCAG TGCCGCCGCG CGCCACGCCCTGCCCGCTTA ACTTAATACC CGCTGTGGTC ACCGCGCCGC  lacZ------------------------------------------------------.  F  Q  F   N  I  S   R  Y  S  Q   Q  Q  L   M  E  T 5251 ACTTCCAGTTCAACATCAGC CGCTACACTC AACAGCAACT GATCGAAACC TCAAGCTCAA GTTGTAGTCGCCGATGTCAG TTGTCGTTCA CTACCTTTGG  lacZ------------------------------------------------------ S  H  R  H   L  L  H   A  E  E   G  T  W  L   N  I  D . 5301 AGCCATCGCCATCTGCTGCA CGCGGAAGAA GGCACATGGC TGAATATCGA TCGGTAGCGG TAGACGACGTGCGCCTTCTT CCGTGTACCG ACTTATAGCT  lacZ------------------------------------------------------. G  F  H   M  G  I  G   G  D  D   S  W  S   P  S  V  S . 5351CGGTTTCCAT ATGGGGATTG GTGGCGACGA CTCCTGGAGC CCGTCAGTAT GCCAAAGGTATACCCCTAAC CACCGCTGCT GAGGACCTCG GGCAGTCATA  lacZ------------------------------------------------------.  A  E  F   Q  L  S   A  G  R  Y   H  Y  Q   L  V  W 5401 CGGCGGAATTCCAGCTGAGC GCCGGTCGCT ACCATTACCA GTTGGTCTGG GCCGCCTTAA GGTCGACTCGCGGCCAGCGA TGGTAATGGT CAACCAGACC   lacZ      AttR2 ---------       -------------------------  C  Q  K 5451 TGTCAAAAAT AATGACTGCAGGTCGACCAT AGTGACTGGA TATGTTGTGT ACAGTTTTTA TTACTGACGT CCAGCTGGTATCACTGACCT ATACAACACA AttR2------------------------------------------------------ 5501 TTTACAGTATTATGTAGTCT GTTTTTTATG CAAAATCTAA TTTAATATAT AAATGTCATA ATACATCAGACAAAAAATAC GTTTTAGATT AAATTATATA AttR2------------------------------------------------------ 5551 TGATATTTATATCATTTTAC GTTTCTCGTT CAGCTTTCTT GTACAAAGTG ACTATAAATA TAGTAAAATGCAAAGAGCAA GTCGAAAGAA CATGTTTCAC AttR2  V5/His -- -------------------------------   G  K  P   I  P  N  P   L  L  G . 5601GTGAGAATGA ATGAAGATCT GGGGAAGCCT ATCCCTAACC CTCTCCTCGG CACTCTTACTTACTTCTAGA CCCCTTCGGA TAGGGATTGG GAGAGGAGCC       V5/His--------------------------------------------.  L  D  S   T  R  T  G   H  H  H   H  H  H 5651 TCTCGATTCT ACGCGTACCGGTCATCATCA CCATCACCAT TGA AGAGCTAAGA TGCGCATGGC CAGTAGTAGT GGTAGTGGTAACT

[1246] TABLE 14 Nucleotide sequence of the Mel/V5-His DEST cassette. phpromoter ----- 1 ATAAGTATTT TACTGTTTTC GTAACAGTTT TGTAATAAAA AAACCTATAATATTCATAAA ATGACAAAAG CATTGTCAAA ACATTATTTT TTTGGATATT 51 ATATTCCGGATTATTCATAC CGTCCCACCA TCGGGCGCGG ATCCTATAAA TATAAGGCCT AATAAGTATGGCAGGGTGGT AGCCCGCGCC TAGGATATTT        Melittin signal -----------------------------------------------------  M  K  F   L  V  N  V   A  L  V   F  M  V   V  Y  I  S . 101 TATGAAATTCTTAGTCAACG TTGCCCTTGT TTTTATGGTC GTATACATTT ATACTTTAAG AATCAGTTGCAACGGGAACA AAAATACCAG CATATGTAAA Melittin signal       attR1---------------          ----------------------- .  Y  I  Y   A 151CTTACATCTA TGCGGCATGG TCGAATCAAA CAAGTTTGTA CAAAAAAGCT GAATGTAGATACGCCGTACC AGCTTAGTTT GTTCAAACAT GTTTTTTCGA attR1------------------------------------------------------ 201 GAACGAGAAACGTAAAATGA TATAAATATC AATATATTAA ATTAGATTTT CTTGCTCTTT GCATTTTACTATATTTATAG TTATATAATT TAATCTAAAA attR1 ----------------------------- 251GCATAAAAAA CAGACTACAT AATACTGTAA AACACAACAT ATCCAGTCAC CGTATTTTTTGTCTGATGTA TTATGACATT TTGTGTTGTA TAGGTCAGTG 301 TATGGCGGCC GCTCCCTAACCCACGGGGCC CGTGGCTATG GCAGGGCTTG ATACCGCCGG CGAGGGATTG GGTGCCCCGGGCACCGATAC CGTCCCGAAC 351 CCGCCCCGAC GTTGGCTGCG AGCCCTGGGC CTTCACCCGAACTTGGGGGT GGCGGGGCTG CAACCGACGC TCGGGACCCG GAAGTGGGCT TGAACCCCCA 401TGGGGTGGGG AAAAGGAAGA AACGCGGGCG TATTGGTCCC AATGGGGTCT ACCCCACCCCTTTTCCTTCT TTGCGCCCGC ATAACCAGGG TTACCCCAGA 451 CGGTGGGGTA TCGACAGAGTGCCAGCCCTG GGACCGAACC CCGCGTTTAT GCCACCCCAT AGCTGTCTCA CGGTCGGGACCCTGGCTTGG GGCGCAAATA 501 GAACAAACGA CCCAACACCC GTGCGTTTTA TTCTGTCTTTTTATTGCCGT CTTGTTTGCT GGGTTGTGGG CACGCAAAAT AAGACAGAAA AATAACGGCA 551CATAGCGCGG GTTCCTTCCG GTATTGTCTC CTTCCGTGTT TCAGTTAGCC GTATCGCGCCCAAGGAAGGC CATAACAGAG GAAGGCACAA AGTCAATCGG    ---    tk gene    N  A  E . 601 TCCCCCATCT CCCGGGCAAA CGTGCGCGCC AGGTCGCAGA +L,32TCGTCGGTAT AGGGGGTAGA GGGCCCGTTT GCACGCGCGG TCCAGCGTCT AGCAGCCATA------------------------------------------------------           tk gene.. G  M  E   R  A  F   T  R  A  L   D  C  I   T  P  I 651 GGAGCCTGGGGTGGTGACGT GGGTCTGGAC CATCCCGGAG GTAAGTTGCA CCTCGGACCC CACCACTGCACCCAGACCTG GTAGGGCCTC CATTCAACGT------------------------------------------------------           tk gene S  G  P  T   T  V  H   T  Q  V   M  G  S  T   L  Q  L . 701 GCAGGGCGTCCCGGCAGCCG GCGGGCGATT GGTCGTAATC CAGGATAAAG CGTCCCGCAG GGCCGTCGGCCGCCCGCTAA CCAGCATTAG GTCCTATTTC------------------------------------------------------           tk gene. L  A  D   R  C  G  A   P  S  Q   D  Y  D   L  I  F  V . 751 ACATGCATGGGACGGAGGCG TTTGGCCAAG ACGTCCAAAG CCCAGGCAAA TGTACGTACC CTGCCTCCGCAAACCGGTTC TGCAGGTTTC GGGTCCGTTT------------------------------------------------------           tk gene.. H  M  P   R  L  R   K  A  L  V   D  L  A   W  A  F 801 CACGTTATACAGGTCGCCGT TGGGGGCCAG CAACTCGGGG GCCCGAAACA GTGCAATATG TCCAGCGGCAACCCCCGGTC GTTGAGCCCC CGGGCTTTGT------------------------------------------------------           tk gene V  N  Y  L   D  G  N   P  A  L   L  E  P  A   R  F  L . 851 GGGTAAATAACGTGTCCCCG ATATGGGGTC GTGGGCCCGC GTTGCTCTGG CCCATTTATT GCACAGGGGCTATACCCCAG CACCCGGGCG CAACGAGACC------------------------------------------------------           tk gene. T  F  L   T  D  G  I   H  P  R   P  G  A   N  S  Q  P . 901 GGCTCGGCACCCTGGGGCGG CACGGCCGCC CCCGAAAGCT GTCCCCAATC CCGAGCCGTG GGACCCCGCCGTGCCGGCGG GGGCTTTCGA CAGGGGTTAG------------------------------------------------------           tk gene.. E  A  G   Q  P  P   V  A  A  G   S  L  Q   G  W  D 951 CTCCCGCCACGACCCGCCGC CCTGCAGATA CCGCACCGTA TTGGCAAGCA GAGGGCGGTG CTGGGCGGCGGGACGTCTAT GGCGTGGCAT AACCGTTCGT------------------------------------------------------           tk gene E  R  W  S   G  G  G   Q  L  Y   R  V  T  N   A  L  L . 1001 GCCCATAAACGCGGCGAATC GCGGCCAGCA TAGCCAGGTC AAGCCGCTCG CGGGTATTTG CGCCGCTTAGCGCCGGTCGT ATCGGTCCAG TTCGGCGAGC------------------------------------------------------           tk gene. G  Y  V   R  R  I  A   A  L  M   A  L  D   L  R  E  G . 1051CCGGGGCGCT GGCGTTTGGC CAGGCGGTCG ATGTGTCTGT CCTCCGGAAG GGCCCCGCGACCGCAAACCG GTCCGCCAGC TACACAGACA GGAGGCCTTC------------------------------------------------------           tk gene.. P  R  Q   R  K  A   L  R  D  I   H  R  D   E  P  L 1101 GGCCCCCAACACGATGTTTG TGCCGGGCAA GGTCGGCGGG ATGAGGGCCA CCGGGGGTTG TGCTACAAACACGGCCCGTT CCAGCCGCCC TACTCCCGGT------------------------------------------------------           tk gene A  G  L  V   I  N  T   G  P  L   T  P  P  I   L  A  V . 1151 CGAACGCCAGCACGGCCTGG GGGGTCATGC TGCCCATAAG GTATCGCGCG GCTTGCGGTC GTGCCGGACCCCCCAGTACG ACGGGTATTC CATAGCGCGC------------------------------------------------------           tk gene. F  A  L   V  A  Q  P   T  M  S   G  M  L   Y  R  A  A . 1201GCCGGGTAGC ACAGGAGGGC GGCGATGGGA TGGCGGTCGA AGATGAGGGT CGGCCCATCGTGTCCTCCCG CCGCTACCCT ACCGCCAGCT TCTACTCCCA------------------------------------------------------           tk gene.. P  Y  C   L  L  A   A  I  P  H   R  D  F   I  L  T 1251 GAGGGCCGGGGGCGGGGCAT GTGAGCTCCC AGCCTCCCCC CCGATATGAG CTCCCGGCCC CCGCCCCGTACACTCGAGGG TCGGAGGGGG GGCTATACTC------------------------------------------------------           tk gene L  A  P  P   P  A  H   S  S  G   A  E  G  G   I  H  P . 1301 GAGCCAGAACGGCGTCGGTC ACGGCATAAG GCATGCCCAT TGTTATCTGG CTCGGTCTTG CCGCAGCCAGTGCCGTATTC CGTACGGGTA ACAATAGACC------------------------------------------------------           tk gene. A  L  V   A  D  T  V   A  Y  P   M  G  M   T  I  Q  A . 1351GCGCTTGTCA TTACCACCGC CGCGTCCCCG GCCGATATCT CACCCTGGTC CGCGAACAGTAATGGTGGCG GCGCAGGGGC CGGCTATAGA GTGGGACCAG------------------------------------------------------           tk gene.. S  T  M   V  V  A   A  D  G  A   S  I  E   G  Q  D 1401 GAGGCGGTGTTGTGTGGTGT AGATGTTCGC GATTGTCTCG GAAGCCCCCA CTCCGCCACA ACACACCACATCTACAAGCG CTAACAGAGC CTTCGGGGGT------------------------------------------------------           tk gene L  R  H  Q   T  T  Y   I  N  A   I  T  E  S   A  G  L . 1451 ACACCCGCCAGTAAGTCATC GGCTCGGGTA CGTAGACGAT ATCGTCGCGC TGTGGGCGGT CATTCAGTAGCCGAGCCCAT GCATCTGCTA TAGCAGCGCG------------------------------------------------------           tk gene. V  R  W   Y  T  M  P   E  P  V   Y  V  I   D  D  R  S . 1501GAACCCAGGG CCACCAGCAG TTGCGTGGTG GTGGTTTTCC CCATCCCGTG CTTGGGTCCCGGTGGTCGTC AACGCACCAC CACCAAAAGG GGTAGGGCAC------------------------------------------------------           tk gene.. G  L  A   V  L  L   Q  T  T  T   T  K  G   M  G  H 1551 GGGACCGTCTATATAAACCC GCAGTAGCGT GGGCATTTTC TGCTCCAGGC CCCTGGCAGA TATATTTGGGCGTCATCGCA CCCGTAAAAG ACGAGGTCCG------------------------------------------------------           tk gene P  G  D  I   Y  V  R   L  L  T   P  M  K  Q   E  L  R . 1601 GGACTTCCGTGGCTTTTTGT TGCCGGCGAG GGCGCAACGC CGTACGTCGG CCTGAAGGCA CCGAAAAACAACGGCCGCTC CCGCGTTGCG GCATGCAGCC------------------------------------------------------           tk gene. V  E  T   A  K  Q  Q   R  R  P   R  L  A   T  R  R  N . 1651TTGTTATGGC CGCGAGAACG CGCAGCCTGG TCGAACGCAG ACGCGTGTTG AACAATACCGGCGCTCTTGC GCGTCGGACC AGCTTGCGTC TGCGCACAAC------------------------------------------------------           tk gene.. N  H  G   R  S  R   A  A  Q  D   F  A  S   A  H  Q 1701 ATGGCAGGGGTACGAAGCCA TAGATCCCGT TATCAATTAC TTATACTATC TACCGTCCCC ATGCTTCGGTATCTAGGGCA ATAGTTAATG AATATGATAG -----------------------        tk gene         ie-0   pr  H  C  P  Y   S  A  M 1751 CGGCGCGCAA GCGAGCGTGTGCGCCGGAGC ACAATTGATA CTGATTTACG GCCGCGCGTT CGCTCGCACA CGCGGCCTCGTGTTAACTAT GACTAAATGC------------------------------------------------------           ie-0 pr1801 AGTTGGGCAA ACGGGCTTTA TATAGCCTGT CCCCTCCACA GCCCTAGTGC TCAACCCGTTTGCCCGAAAT ATATCGGACA GGGGAGGTGT CGGGATCACG------------------------------------------------------           ie-0 pr1851 CGTGCGCAAA GTGCCTACGT GACCAGGCTC TCCTACGCAT ATACAATCTT GCACGCGTTTCACGGATGCA CTGGTCCGAG AGGATGCGTA TATGTTAGAA------------------------------------------------------           ie-0 pr1901 ATCTCTATAG ATAAGGTTTC CATATATAAA GCCTCTCGAT GGCTGAACGT TAGAGATATCTATTCCAAAG GTATATATTT CGGAGAGCTA CCGACTTGCA------------------------------------------------------           ie-0 pr1951 GCACAGTATC GTGTTGATTT CTGAGTGCTA ACTAACAGTT ACAATGAACC CGTGTCATAGCACAACTAAA GACTCACGAT TGATTGTCAA TGTTACTTGG------------------------------------------------------           ie-0 pr2001 GTTTTTTTCG AGAGAATAAC ATTTTTGACG CGCCAAGGAC CGGGGGCAAG CAAAAAAAGCTCTCTTATTG TAAAAACTGC GCGGTTCCTG GCCCCCGTTC------------------------------------------------------           ie-0 pr2051 GGTCGTGCCA AATCTTTGCC AGCGCCTGCC GCCAACTCGC CGCCGTCGCC CCAGCACGGTTTAGAAACGG TCGCGGACGG CGGTTGAGCG GCGGCAGCGG------------------------------------------------------           ie-0 pr2101 TGTTCGTCCG CCGCCAAAAT CTAACATCAA ACCACCTACG CGCATCTCTC ACAAGCAGGCGGCGGTTTTA GATTGTAGTT TGGTGGATGC GCGTAGAGAG------------------------------------------------------           ie-0 pr2151 CGCCTAAACA GCCTATGTGC ACCTCTCCGG CCAAGCCGTT GGAGCACAGC GCGGATTTGTCGGATACACG TGGAGAGGCC GGTTCGGCAA CCTCGTGTCG------------------------------------------------------           ie-0 pr2201 AGCATTGTAA GTAAAAAACC AGTCGTCAAC AGAAAAGATG GATATTTTGT TCGTAACATTCATTTTTTGG TCAGCAGTTG TCTTTTCTAC CTATAAAACA------------------------------------------------------           ie-0 pr2251 GCCGCCCGAG TTTGGGAACA AGTTTGAAGG TTTGCCCGCG TACAGCGACA CGGCGGGCTCAAACCCTTGT TCAAACTTCC AAACGGGCGC ATGTCGCTGT------------------------------------------------------           ie-0 pr        p10   pr         -- 2301 AACTGGATTT CAAACAAGAG CGCGATCTACGTACCTGCAG GCCCGGGCTC TTGACCTAAA GTTTGTTCTC GCGCTAGATG CATGGACGTCCGGGCCCGAG --------------------------------------------      ie-0 pr          p10 pr ------------------------------------------------------2351 AACCCAACAC AATATATTAT AGTTAAATAA GAATTATTAT CAAATCATTT TTGGGTTGTGTTATATAATA TCAATTTATT CTTAATAATA GTTTAGTAAA          p10 pr--------------------------------------------- 2401 GTATATTAAT TAAAATACTATACTGTAAAT TACATTTTAT TTACAATTCA CATATAATTA ATTTTATGAT ATGACATTTAATGTAAAATA AATGTTAAGT     lacZ       -----------------------------------------------        M   T  M  I  T   D  S  L   A  V  V   L  Q  R  R . 2451CTCTAGAATG ACCATGATTA CGGATTCACT GGCCGTCGTT TTACAACGTC GAGATCTTACTGGTACTAAT GCCTAAGTGA CCGGCAGCAA AATGTTGCAG  lacZ------------------------------------------------------.  D  W  E   N  P  G   V  T  Q  L   N  R  L   A  A  H 2501 GTGACTGGGAAAACCCTGGC GTTACCCAAC TTAATCGCCT TGCAGCACAT CACTGACCCT TTTGGGACCGCAATGGGTTG AATTAGCGGA ACGTCGTGTA  lacZ------------------------------------------------------ P  P  F  A   S  W  R   N  S  E   E  A  R  T   D  R  P . 2551 CCCCCTTTCGCCAGCTGGCG TAATAGCGAA GAGGCCCGCA CCGATCGCCC GGGGGAAAGC GGTCGACCGCATTATCGCTT CTCCGGGCGT GGCTAGCGGG  lacZ------------------------------------------------------. S  Q  Q   L  R  S  L   N  G  E   W  R  F   A  W  F  P . 2601TTCCCAACAG TTGCGCAGCC TGAATGGCGA ATGGCGCTTT GCCTGGTTTC AAGGGTTGTCAACGCGTCGG ACTTACCGCT TACCGCGAAA CGGACCAAAG  lacZ------------------------------------------------------     Bsu36I    ------ .  A  P  E   A  V  P   E  S  W  L   E  C  D   L  P  E 2651CGGCACCAGA AGCGGTGCCG GAAAGCTGGC TGGAGTGCGA TCTTCCTGAG GCCGTGGTCTTCGCCACGGC CTTTCGACCG ACCTCACGCT AGAAGGACTC  lacZ------------------------------------------------------ Bsu36I - A  D  T  V   V  V  P   S  N  W   Q  M  H  G   Y  D  A . 2701 GCCGATACTGTCGTCGTCCC CTCAAACTCG CAGATGCACG GTTACGATGC CGGCTATGAC AGCAGCAGGGGAGTTTCACC GTCTACGTGC CAATGCTACG  lacZ------------------------------------------------------. P  I  Y   T  N  V  T   Y  P  I   T  V  N   P  P  F  V . 2751GCCCATCTAC ACCAACCTAA CCTATCCCAT TACGGTCAAT CCGCCGTTTG CGGGTAGATGTGGTTGCATT GGATAGGGTA ATGCCAGTTA GGCGGCAAAC  lacZ------------------------------------------------------.  P  T  E   N  P  T   G  C  Y  S   L  T  F   N  V  D 2801 TTCCCACCGAGAATCCGACG GGTTGTTACT CGCTCACATT TAATGTTGAT AAGGGTGCCT CTTAGGCTGCCCAACAATGA GCGAGTGTAA ATTACAACTA  lacZ------------------------------------------------------ E  S  W  L   Q  E  C   Q  T  R   I  I  F  D   G  V  N . 2851 GAAAGCTGGCTACAGGAAGG CCAGACGCCA ATTATTTTTG ATGGCGTTAA CTTTCGACCG ATGTCCTTCCGGTCTGCCCT TAATAAAAAC TACCCCAATT  lacZ------------------------------------------------------. S  A  F   H  L  W  C   N  G  R   W  V  G   Y  G  Q  D . 2901CTCGGCGTTT CATCTGTGGT GCAACGGGCG CTGGGTCGGT TACCGCCAGG GAGCCGCAAAGTAGACACCA CCTTGCCCCC CACCCAGCCA ATCCCCCTCC  lacZ------------------------------------------------------.  S  R  L   P  S  E   F  D  L  S   A  F  L   R  A  G 2951 ACACTCGTTTGCCGTCTGAA TTTGACCTCA GCCCATTTTT ACGCGCCGGA TCTCACCAAA CGCCAGACTTAAACTGGACT CCCGTAAAAA TCCGCGGCCT  lacZ------------------------------------------------------ E  N  R  L   A  V  M   V  L  R   W  S  D  G   S  Y  L . 3001 GAAAACCCCCTCCCCCTCAT GCTGCTCCCT TCCAGTCACC CCAGTTATCT CTTTTGGCCC AGCGCCACTACCACCACCCA ACCTCACTCC CCTCAATACA  lacZ------------------------------------------------------. E  D  Q   D  M  W  R   M  S  G   I  F  R   D  V  S  L . 3051CCAAGATCAC GATATCTCGC CCATCACCGG CATTTTCCCT CACCTCTCGT CCTTCTACTCCTATACACCG CCTACTCCCC GTAAAAGGCA CTCCACACCA  lacZ------------------------------------------------------.  L  H  K   P  T  T   Q  I  S  D   F  H  V   A  T  R 3101 TCCTCCATAAACCGACTACA CAAATCAGCC ATTTCCATCT TCCCACTCGC ACCACCTATT TGCCTCATCTGTTTACTCCC TAAACCTACA ACCGTCAGCC  lacZ------------------------------------------------------ F  N  D  D   F  S  R   A  V  L   E  A  E  V   Q  M  C . 3151 TTTAATGATGATTTCAGCCG CGCTGTACTG GAGGCTGAAG TTCAGATGTG AAATTACTAC TAAAGTCGGCGCGACATGAC CTCCGACTTC AAGTCTACAC  lacZ------------------------------------------------------. G  E  L   R  D  Y  L   R  V  T   V  S  L   W  Q  G  E . 3201CGGCGAGTTG CGTGACTACC TACGGGTAAC AGTTTCTTTA TGGCAGGGTG GCCGCTCAACGCACTGATGG ATGCCCATTG TCAAAGAAAT ACCGTCCCAC  lacZ------------------------------------------------------.  T  Q  V   A  S  G   T  A  P  F   G  G  E   I  I  D 3251 AAACGCAGGTCGCCAGCGGC ACCGCGCCTT TCGGCGGTGA AATTATCGAT TTTGCGTCCA GCGGTCGCCGTGGCGCGGAA AGCCGCCACT TTAATAGCTA  lacZ------------------------------------------------------ E  R  G  G   Y  A  D   R  V  T   L  R  L  N   V  E  N . 3301 GAGCGTGGTGGTTATGCCGA TCGCGTCACA CTACGTCTGA ACGTCGAAAA CTCGCACCAC CAATACGGCTAGCGCAGTGT GATGCAGACT TGCAGCTTTT  lacZ------------------------------------------------------. P  K  L   W  S  A  E   I  P  N   L  Y  R   A  V  V  E . 3351CCCGAAACTG TGGAGCGCCG AAATCCCGAA TCTCTATCGT GCGGTGGTTG GGGCTTTGACACCTCGCGGC TTTAGGGCTT AGAGATAGCA CGCCACCAAC  lacZ------------------------------------------------------.  L  H  T   A  D  G   T  L  I  E   A  E  A   C  D  V 3401 AACTGCACACCGCCGACGGC ACGCTGATTG AAGCAGAAGC CTGCGATGTC TTGACGTGTG GCGGCTGCCGTGCGACTAAC TTCGTCTTCG GACGCTACAG  lacZ------------------------------------------------------ G  F  R  E   V  R  I   E  N  G   L  L  L  L   N  G  K . 3451 GGTTTCCGCGAGGTGCGGAT TGAAAATGGT CTGCTGCTGC TGAACGGCAA CCAAAGGCGC TCCACCCCTAACTTTTACCA GACGACGACG ACTTGCCGTT  lacZ------------------------------------------------------. P  L  L   I  R  G  V   N  R  H   E  H  H   P  L  H  G . 3501GCCGTTGCTG ATTCCAGGCG TTAACCGTCA CGAGCATCAT CCTCTGCATG CCGCAACCACTAAGCTCCGC AATTGGCACT CCTCGTACTA GGAGACGTAC  lacZ------------------------------------------------------.  Q  V  M   D  E  Q   T  M  V  Q   D  I  L   L  M  K 3551 GTCAGGTCATCGATGACCAC ACGATGGTGC ACGATATCCT GCTGATGAAG CAGTCCAGTA CCTACTCGTCTGCTACCACG TCCTATAGGA CGACTACTTC  lacZ------------------------------------------------------ Q  N  N  F   N  A  V   R  C  S   H  Y  P  N   H  P  L . 3601 CAGAACAACTTTAACGCCGT GCGCTGTTCG CATTATCCGA ACCATCCGCT GTCTTGTTGA AATTGCGGCACGCGACAAGC GTAATAGGCT TGGTAGGCGA  lacZ------------------------------------------------------. W  Y  T   L  C  D  R   Y  G  L   Y  V  V   D  E  A  N . 3651GTGGTACACG CTGTGCGACC GCTACGGCCT GTATGTGGTG GATGAAGCCA CACCATGTGCGACACGCTGG CGATGCCGGA CATACACCAC CTACTTCGGT  lacZ------------------------------------------------------.  I  E  T   H  G  M   V  P  M  N   R  L  T   D  D  P 3701 ATATTGAAACCCACGGCATG GTGCCAATGA ATCGTCTGAC CGATGATCCG TATAACTTTG GGTGCCGTACCACGGTTACT TAGCAGACTG GCTACTAGGC  lacZ------------------------------------------------------ R  W  L  P   A  M  S   E  R  V   T  R  M  V   Q  R  D . 3751 CGCTGGCTACCGGCGATGAG CGAACGCGTA ACGCGAATGG TGCAGCGCGA GCGACCGATG GCCGCTACTCGCTTGCGCAT TGCGCTTACC ACGTCGCGCT  lacZ------------------------------------------------------. R  N  H   P  S  V  I   I  W  S   L  G  N   E  S  G  H . 3801TCGTAATCAC CCGAGTGTGA TCATCTGGTC GCTGGGGAAT GAATCAGGCC AGCATTAGTGGGCTCACACT AGTAGACCAG CGACCCCTTA CTTAGTCCGG  lacZ------------------------------------------------------.  G  A  N   H  D  A   L  Y  R  W   I  K  S   V  D  P 3851 ACGGCGCTAATCACGACGCG CTGTATCGCT GGATCAAATC TGTCGATCCT TGCCGCGATT AGTGCTGCGCGACATAGCGA CCTAGTTTAG ACAGCTAGGA  lacZ------------------------------------------------------ S  R  P  V   Q  Y  E   G  G  G   A  D  T  T   A  T  D . 3901 TCCCGCCCGGTGCAGTATGA AGGCGGCGGA GCCGACACCA CGGCCACCGA AGGGCGGGCC ACGTCATACTTCCGCCGCCT CGGCTGTGGT GCCGGTGGCT  lacZ------------------------------------------------------. I  I  C   P  M  Y  A   R  V  D   E  D  Q   P  F  P  A . 3951TATTATTTGC CCGATGTACG CGCGCGTGGA TGAAGACCAG CCCTTCCCGG ATAATAAACGGGCTACATGC GCGCGCACCT ACTTCTGGTC GGGAAGGGCC  lacZ------------------------------------------------------.  V  P  K   W  S  I   K  K  W  L   S  L  P   G  E  T 4001 CTGTGCCGAAATGGTCCATC AAAAAATGGC TTTCGCTACC TGGAGAGACG GACACGGCTT TACCAGGTAGTTTTTTACCG AAAGCGATGG ACCTCTCTGC  lacZ------------------------------------------------------ R  P  L  I   L  C  E   Y  A  H   A  M  G  N   S  L  G . 4051 CGCCCGCTGATCCTTTGCGA ATACGCCCAC GCGATGGGTA ACAGTCTTGG GCGGGCGACT AGGAAACGCTTATGCGGGTG CGCTACCCAT TGTCAGAACC  lacZ------------------------------------------------------. G  F  A   K  Y  W  Q   A  F  R   Q  Y  P   R  L  Q  G . 4101CGGTTTCGCT AAATACTGGC AGGCGTTTCG TCAGTATCCC CGTTTACAGG GCCAAAGCGATTTATGACCG TCCGCAAAGC AGTCATAGGG GCAAATGTCC  lacZ------------------------------------------------------.  G  F  V   W  D  W   V  D  Q  S   L  I  K   Y  D  E 4151 GCGGCTTCGTCTGGGACTGG GTGGATCAGT CGCTGATTAA ATATGATGAA CGCCGAAGCA GACCCTGACCCACCTAGTCA GCGACTAATT TATACTACTT  lacZ------------------------------------------------------ N  G  N  P   W  S  A   Y  G  G   D  F  G  D   T  P  N . 4201 AACGGCAACCCGTGGTCGGC TTACGGCGGT GATTTTGGCG ATACGCCGAA TTGCCGTTGG GCACCAGCCGAATGCCGCCA CTAAAACCGC TATGCGGCTT  lacZ------------------------------------------------------. D  R  Q   F  C  M  N   G  L  V   F  A  D   R  T  P  H . 4251CGATCGCCAG TTCTGTATGA ACGGTCTGGT CTTTGCCGAC CGCACGCCGC GCTAGCGGTCAAGACATACT TGCCAGACCA GAAACGGCTG GCGTGCGGCG  lacZ------------------------------------------------------.  P  A  L   T  E  A   K  H  Q  Q   Q  F  F   Q  F  R 4301 ATCCAGCGCTGACGGAAGCA AAACACCAGC AGCAGTTTTT CCAGTTCCGT TAGGTCGCGA CTGCCTTCGTTTTGTGGTCG TCGTCAAAAA GGTCAAGGCA  lacZ------------------------------------------------------ L  S  G  Q   T  I  E   V  T  S   E  Y  L  F   R  H  S . 4351 TTATCCGGGCAAACCATCGA AGTGACCAGC GAATACCTCT TCCGTCATAG AATAGGCCCG TTTCGTACCTTCACTGGTCC CTTATGGACA AGGCAGTATC  lacZ------------------------------------------------------. D  N  E   L  L  H  W   M  V  A   L  D  G   K  P  L  A . 4401CGATAACGAG CTCCTGCACT GCATGGTGGC CCTGGATGGT AAGCCGCTGG GCTATTGCTCGACCACGTGA CCTACCACCG CCACCTACCA TTCGGCGACC  lacZ------------------------------------------------------.  S  G  E   V  P  L   D  V  A  P   Q  G  K   Q  L  I 4451 CAAGCGGTGAAGTGCCTCTG GATGTCGCTC CACAAGGTAA ACAGTTGATT GTTCGCCACT TCACGGAGACCTACAGCGAG GTGTTCCATT TGTCAACTAA  lacZ------------------------------------------------------ E  L  P  E   L  P  Q   P  E  S   A  G  Q  L   W  L  T . 4501 GAACTGCCTGAACTACCGCA GCCGGAGAGC GCCGGGCAAC TCTGGCTCAC CTTGACGGAC TTGATGGCGTCGGCCTCTCG CGGCCCGTTG AGACCGAGTG  lacZ------------------------------------------------------. V  R  V   V  Q  P  N   A  T  A   W  S  E   A  G  H  I . 4551AGTACGCGTA GTGCAACCGA ACGCGACCGC ATGGTCAGAA GCCGGGCACA TCATGCGCATCACGTTGGCT TGCGCTGGCG TACCAGTCTT CGGCCCGTGT  lacZ------------------------------------------------------.  S  A  W   Q  Q  W   R  L  A  E   N  L  S   V  T  L 4601 TCAGCGCCTGGCAGCAGTGG CGTCTGGCGG AAAACCTCAG TGTGACGCTC AGTCGCGGAC CGTCGTCACCGCAGACCGCC TTTTGGAGTC ACACTGCGAG  lacZ------------------------------------------------------ P  A  A  S   H  A  I   P  H  L   T  T  S  E   M  D  F . 4651 CCCGCCGCGTCCCACGCCAT CCCGCATCTG ACCACCAGCG AAATGGATTT GGGCGGCGCA GGGTGCGGTAGGGCGTAGAC TGGTGGTCGC TTTACCTAAA  lacZ------------------------------------------------------. C  I  E   L  G  N  K   R  W  Q   F  N  R   Q  S  G  F . 4701TTGCATCGAG CTGGGTAATA AGCGTTGGCA ATTTAACCGC CAGTCAGGCT AACGTAGCTCGACCCATTAT TCGCAACCGT TAAATTGGCG GTCAGTCCGA  lacZ------------------------------------------------------.  L  S  Q   M  W  I   G  D  K  K   Q  L  L   T  P  L 4751 TTCTTTCACAGATGTGGATT GGCGATAAAA AACAACTGCT GACGCCGCTG AAGAAAGTGT CTACACCTAACCGCTATTTT TTGTTGACGA CTGCGGCGAC  lacZ------------------------------------------------------ R  D  Q  F   T  R  A   P  L  D   N  D  I  G   V  S  E . 4801 CGCGATCAGTTCACCCGTGC ACCGCTGGAT AACGACATTG GCGTAAGTGA GCGCTAGTCA AGTGGGCACGTGGCGACCTA TTGCTGTAAC CGCATTCACT  lacZ------------------------------------------------------. A  T  R   I  D  P  N   A  W  V   E  R  W   K  A  A  G . 4851AGCGACCCGC ATTGACCCTA ACGCCTGGGT CGAACGCTGG AAGGCGGCGG TCGCTGGGCGTAACTGGGAT TGCGGACCCA GCTTGCGACC TTCCGCCGCC  lacZ------------------------------------------------------.  H  Y  Q   A  E  A   A  L  L  Q   C  T  A   D  T  L 4901 GCCATTACCAGGCCGAAGCA GCGTTGTTGC AGTGCACGGC AGATACACTT CGGTAATGGT CCGGCTTCGTCGCAACAACG TCACGTGCCG TCTATGTGAA  lacZ------------------------------------------------------ A  D  A  V   L  I  T   T  A  H   A  W  Q  H   Q  G  K . 4951 CCTGATGCGGTCCTGATTAC GACCGCTCAC GCGTGGCACC ATCAGGGGAA CGACTACGCC ACGACTAATGCTGCCGAGTG CGCACCGTCG TAGTCCCCTT  lacZ------------------------------------------------------. T  L  F   I  S  R  K   T  Y  R   I  D  G   S  G  Q  M . 5001AACCTTATTT ATCAGCCGGA AAACCTACCG GATTGATCGT AGTGGTCAAA TTGCAATAAATAGTCGGCCT TTTGGATGGC CTAACTACCA TCACCAGTTT  lacZ------------------------------------------------------.  A  I  T   V  D  V   E  V  A  S   D  T  P   H  P  A 5051 TCGCGATTACCGTTGATGTT GAAGTGGCGA CCGATACACC GCATCCGGCG ACCGCTAATG GCAACTACAACTTCACCGCT CGCTATGTGG CGTAGCCCGC  lacZ------------------------------------------------------ R  I  G  L   N  C  Q   L  A  Q   V  A  E  R   V  N  W . 5101 CGGATTGGCCTGAACTGCCA CCTGGCGCAG GTAGCAGAGC GGGTAAACTG GCCTAACCGG ACTTGACGGTCGACCGCGTC CATCGTCTCG CCCATTTGAC  lacZ------------------------------------------------------. L  G  L   G  P  Q  E   N  Y  P   D  R  L   T  A  A  C . 5151GCTCGGATTA GGCCCGCAAG AAAACTATCC CGACCGCCTT ACTGCCGCCT CGAGCCTAATCCCGGCGTTC TTTTGATAGC GCTGGCGGAA TGACGGCCGA  lacZ------------------------------------------------------.  F  D  R   W  D  L   P  L  S  D   M  Y  T   P  Y  V 5201 GTTTTGACCGCTCGGATCTG CCATTGTCAG ACATGTATAC CCCGTACGTC CAAAACTCGC CACCGTAGACGGTAACACTC TGTACATATG GGGCATGCAG  lacZ------------------------------------------------------ F  P  S  E   N  G  L   R  C  G   T  R  E  L   N  Y  G . 5251 TTCCCGAGCCAAAACCGTCT GCGCTGCGGG ACGCCCGAAT TGAATTATGG AAGGGCTCGC TTTTGCCAGACCCCACGCCC TGCGCGCTTA ACTTAATACC  lacZ------------------------------------------------------. P  H  Q   W  R  G  D   F  Q  F   N  I  S   R  Y  S  Q . 5301CCCACACCAG TGGCGCGGCG ACTTCCACTT CAACATCAGC CGCTACAGTC GGGTGTCGTCACCCCGCCCC TCAAGCTCAA GTTGTAGTCC GCGATGTCAG  lacZ------------------------------------------------------.  Q  Q  L   M  E  T   S  H  R  H   L  L  H   A  E  E 5351 AACACCAACTGATCCAAACC ACCCATCCCC ATCTCCTCCA CCCCCAACAA TTGTCCTTCA CTACCTTTGGTCCGTACCGC TAGACGACCT GCCCCTTCTT  lacZ------------------------------------------------------ G  T  W  L   N  I  D   G   F  H   M  G  I  G   G  D  D . 5401GGCACATGGC TGAATATCGA CGGTTTCCAT ATGGGGATTG GTGGCGACGA CCGTGTACCGACTTATAGCT GCCAAAGGTA TACCCCTAAC CACCGCTGCT  lacZ------------------------------------------------------. S  W  S   P  S  V  S   A  E  F   Q  L  S   A  G  R  Y . 5451CTCCTGGAGC CCGTCAGTAT CGGCGGAATT CCAGCTGAGC GCCGGTCGCT GAGGACCTCGGGCAGTCATA GCCGCCTTAA GGTCGACTCG CGGCCAGCGA  lacZ        AttR2-------------------------------        ---.  H  Y  Q   L  V  W   C  Q  K 5501 ACCATTACCA GTTGGTCTGG TGTCAAAAATAATGACTGCA GGTCGACCAT TGGTAATGGT CAACCAGACC ACAGTTTTTA TTACTGACGTCCAGCTGGTA AttR2 ------------------------------------------------------5551 AGTGACTGGA TATGTTGTGT TTTACAGTAT TATGTAGTCT GTTTTTTATG TCACTGACCTATACAACACA AAATGTCATA ATACATCAGA CAAAAAATAC AttR2------------------------------------------------------ 5601 CAAAATCTAATTTAATATAT TGATATTTAT ATCATTTTAC GTTTCTCGTT GTTTTAGATT AAATTATATAACTATAAATA TAGTAAAATG CAAAGAGCAA         AttR2   V5/His------------------------  ---------   G  K  P 5651 CAGCTTTCTT GTACAAAGTGGTGAGAATGA ATGAAGATCT GGGGAAGCCT GTCGAAAGAA CATGTTTCAC CACTCTTACTTACTTCTAGA CCCCTTCGGA V5/His------------------------------------------------------ I  P  N  P   L  L  G   L  D  S   T  R  T  G   H  H  H . 5701 ATCCCTAACCCTCTCCTCGG TCTCGATTCT ACGCGTACCG GTCATCATCA TAGGGATTGG GAGAGGAGCCAGAGCTAAGA TGCGCATGGC CAGTAGTAGT stop codon ---   V5/His -----------. H  H  H 5751 CCATCACCAT TGA GGTAGTGGTA ACT

[1247] TABLE 15 Baculoviral promoter sequences. AcMNPV ORF 25 promotersequenceGgtgtcttcattagtatgccaatcacgtacgcaacagtcgcaaaagaaacacacagtttcgtctccgcgacccgtgtaaaaaagtcccgcttccgcaatgtttgtaatcatgtcacgcaatgcggcaggccaaaagttaacaaacgtatccatacgcgactgtaaattggacatgcatctgtacacacacttgggtttgccttctttcactagtacagcgttgatggtaatgttgtcgccaaacgattcacgctcggcgatcttgttagcatacgcgcaatacggcgacaaggttacgtgtgcatattcaatacactcgtcttcggaccaattttttatttctgcttcgcaatactcgcacacaacgtgatcgtcaacttgattgtatttaaacccgttaacgatcaagctgttaataaacgccgtgttttcaatgggataattttcaaacgaactatgtctttctattaacatgtcgaatacgtgttcggcggtgttgtcgcgaaagttgtcacacacgctgataaaataaaacgggggcgtgtcctcgttcattttagctcgttaaagttacggtcaaaatgagcacgtttgcgtcgttttggtttagcgacacgtttatatggcccagtttggtttttgtttcggcgttaatgacgtgcactgtggacaaatcgtgttctaaaactacaaactcgtactcgaaaatgtttgatatgtagttggttagccgatctatcttaaaattaaacttttgcaactcgctgatagagcacacgtccacatacttgtcgataaacccgttgctcaaccgcttcaaaacggtgtaattttgtagcttgaaaggggcgcatttggaatgactaaaaggaatatttttcaataaatcgtcagtagtgtacgcaaacgcgttgtctacgcacatgctggcaacagagtcgtccatatttattatatatcttatattctgtgaaacacttcaattagacttgaaccacagcagacagcgcacgtcggtagc AcMNPV lef 3 promoterCcgagaagaaggcggtttgtataaaacccatttttcgaaatggttaacaaacttgtttagcatttggatcgtttcgtgttcaaacgcgtcgaaaacttttaaaacgcaattgccgccgggacgcaggcaaattaaaattagctgcgtctcgcacatgatcaaatcaaagttgagacgttcttgttcgttttcgcgtccattaacgtcaaccgagccatctgccaacaccagatcgcacgcgttgccacacttgatgctaatctcaaatacaacatttttatcaaacacgtcgcctgacttgtcgggccccgtaatggttgtgaaatttttgcgtttgcgcactgtcggtttgtacacgcacaccgagttgtttgtcaacgtgacgccatacgctttgcaaagcgggttcaacgacatggtatagttggcaaactcgcccggtccgccgcacaaatccaaaaacgtgtcaacgtgtcggcaaacgtgaaactttttgtcgatctctgatagttttcgccaacatctaggtctgcgcgttgggcgtttgtcaaataattttgagcgagcgcaaaccaccgacttgctgctgaacgtgttcaaaccatctttgagtttatttaatttttgctgcaacatttttactcttcgtgtcggtcgcaatgtttgtgtcgaaaaagacggccaacacgctcagcaaaactatacaaataaagaacaaaaatacgtacgcaatattaacattgaccgtttgatcgttaaatcggacgggtctgttcagagccgctcttattctctcgttgtacattgttaaagtttttgtttttaaattgtacacaatcggcgtgttgtagtcgaaattttcaaaatcggctttttgaaacattgttctgaacgtgttgtcgagcggcgtgttgctggccacgtttataatcaactccctccacgctaacgaacggtgctctggcgacacttcgatttcgtcgccattcagtatttgccatcggatagattcccacatatcgacaacagcaat AcMNPV TLP promotertgctagcccaattggccactgttgtacgaaatatcgtcgtcaacgtgtttgaatacatgttggcccgtaccgttgggtaaatctatgcatctggagtcgccggaacactcgtactggttgtcagagtttctgatccggttgatgcacgttatcagttgtgactcgttattattcaaacatttgaaatattgcgtgtcgccgatatcggccgttatgtacgtgtgtccggcgccgttaaacgcgcacggatgcgcttccacgcacgacattaagttgcgatcaaatattttattcgcggggcattcgcccaccacgtggcgcccatttacgcactgcataaactggttgacgagcaaattggagggaaagtatgatagtatatagccgtctggcctgttttcacacaattcgttaactttacactggccggtttccgcgtcaaacgtgtaattatctggacattcttcgactgcgtgcgctccgtttgcaaaacacctaagatagaacgtgggatgatacaagtgcgcgttggtagaataatctttgtccaagtgttggttcaacaccaacgtgtccagcaaacgctcgtccatgggataaagaccggcagacttgttgtcgcacggcggcacgggaacacattttagttgtgcgtaatcaaagttaaaatatgcggggcatttcatggtcacgtcggccttgtcgccgctcaaaataaactcgttgggattttcatcatttgctctaacgcgatcgtgtacgattcgatcaacaggttgaaatttttgatttaagaaatcaaaaatttcaatccggtcatcatgcacgctttcgtgataggtggaaaggtcgacggtgttgaaccacgttacaatataagtgttttgcataatatccgacacgtagcctattacgtcgggtgtgggttcgtctgcgttggtgcgcttcacatattcagtcatcacttggagccgcttggtgaaagtcgtttcgtcaaattcaaaataaattgccaaatacattaaagtaaacgctattataagaaaaaagctt AcMNPV hr5 sequenceGttttacgcgtagaattctacccgtaaagcgagtttagttatgagccatgtgcaaaacatgacatcagcttttatttttataacaaatgacatcatttcttgattgtgttttacacgtagaattctactcgtaaagcgagttcagttttgaaaaacaaatgacatcatctttttgattgtgctttacaagtagaattctacccgtaaatcaagttcggttttgaaaaacaaatgagtcatattgtatgatatcatattgcaaacaaatgactcatcaatcgatcgtgcgtacacgtagaattctactcgtaaagcgagtttatgagccgtgtgcaaaacatgacatcatctcgatttgaaaaacaaatgacatcatccactgatcgtgcgttacaagtagaattctactcgtaaagccagttcggttatgagccgtgtgcaaaacatgacatcagcttatgactcgtacttgattgtgttttacgcgtagaattctactcgtaaagc

[1248] TABLE 16 IE-1 promoter, coding, and polypeptide sequence. AcMNPVIE-1 promoterGttttacgcgtagaattctacccgtaaagcgagtttagttatgagccatgtgcaaaacatgacatcagcttttatttttataacaaatgacatcatttcttgattgtgttttacacgtagaattctactcgtaaagcgagttcagttttgaaaaacaaatgacatcatctttttgattgtgctttacaagtagaattctacccgtaaatcaagttcggttttgaaaaacaaatgagtcatattgtatgatatcatattgcaaacaaatgactcatcaatcgatcgtgcgtacacgtagaattctactcgtaaagcgagtttatgagccgtgtgcaaaacatgacatcatctcgatttgaaaaacaaatgacatcatccactgatcgtgcgttacaagtagaattctactcgtaaagccagttcggttatgagccgtgtgcaaaacatgacatcagcttatgactcgtacttgattgtgttttacgcgtagaattctactcgtaaagc AcMPNV IE-1 coding sequenceatgacgcaaattaattttaacgcgtcgtacaccagcgcttcgacgccgtcccgagcgtcgttcgacaacagctattcagagttttgtgataaacaacccaacgactatttaagttattataaccatcccaccccggatggagccgacacggtgatatctgacagcgagactgcggcagcttcaaactttttggcaagcgtcaactcgttaactgataatgatttagtggaatgtttgctcaagaccactgataatctcgaagaagcagttagttctgcttattattcggaatcccttgagcagcctgttgtggagcaaccatcgcccagttctgcttatcatgcggaatcttttgagcattctgctggtgtgaaccaaccatcggcaactggaactaaacggaagctggacgaatacttggacaattcacaaggtgtggtgggccagtttaacaaaattaaattgaggcctaaatacaagaaaagcacaattcaaagctgtgcaacccttgaacagacaattaatcacaacacgaacatttgcacggtcgcttcaactcaagaaattacgcattattttactaatgattttgcgccgtatttaatgcgtttcgacgacaacgactacaattccaacaggttctccgaccatatgtccgaaactggttattacatgtttgtggttaaaaaaagtgaagtgaagccgtttgaaattatatttgccaagtacgtgagcaatgtggtttacgaatatacaaacaattattacatggtagataatcgcgtgtttgtggtaacttttgataaaattaggtttatgatttcgtacaatttggttaaagaaaccggcatagaaattcctcattctcaagatgtgtgcaacgacgagacggctgcacaaaattgtaaaaaatgccatttcgtcgatgtgcaccacacgtttaaagctgctctgacttcatattttaatttagatatgtattacgcgcaaaccacatttgtgactttgttacaatcgttgggcgaaagaaaatgtgggtttcttttgagcaagttgtacgaaatgtatcaagataaaaatttatttactttgcctattatgcttagtcgtaaagagagtaatgaaattgagactgcatctaataatttctttgtatcgccgtatgtgagtcaaatattaaagtattcggaaagtgtgcagtttcccgacaatcccccaaacaaatatgtggtggacaatttaaatttaattgttaacaaaaaaagtacgctcacgtacaaatacagcagcgtcgctaatcttttgtttaataattataaatatcatgacaatattgcgagtaataataacgcagaaaatttaaaaaaggttaagaaggaggacggcagcatgcacattgtcgaacagtatttgactcagaatgtagataatgtaaagggtcacaattttatagtattgtctttcaaaaacgaggagcgattgactatagctaagaaaaacaaagagttttattggatttctggcgaaattaaagatgtagacgttagtcaagtaattcaaaaatataatagatttaagcatcacatgtttgtaatcggtaaagtgaaccgaagagagagcactacattgcacaataatttgttaaaattgttagctttaatattacagggtctggttccgttgtccgacgctataacgtttgcggaacaaaaactaaattgtaaatataaaaaattcgaatttaatAcMNPV IE-1 protein sequenceMtqinfnasytsastpsrasfdnsysefcdkqpndylsyynhptpdgadtvisdsetaaasnflasvnsltdndlvecllkttdnleeavssayysesleqpvveqpspssayhaesfehsagvnqpsatgtkrkldeyldnsqgvvgqfnkiklrpkykkstiqscatleqtinhntnictvastqeithyftndfapylmrfddndynsnrfsdhmsetgyymfvvkksevkpfeiifakyvsnvvyeytnnyymvdnrvfvvtfdkirfmisynlvketgieiphsqdvcndetaaqnckkchfvdvhhtfkaaltsyfnldmyyaqttfvtllqslgerkcgfllsklyemyqdknlftlpimlsrkesneietasnnffvspyvsqilkysesvqfpdnppnkyvvdnlnlivnkkstltykyssvanllfnnykyhdniasnnnaenlkkvkkedgsmhiveqyltqnvdnvkghnfivlsfkneerltiakknkefywisgeikdvdvsqviqkynrfkhhmfvigkvnrresttlhnnllkllalilqglvplsdaitfaeqklnckykkfefn

[1249] TABLE 17 Nucleotide sequence of plasmid pLenti6/V5-DEST.AATGTAGTCTTATGCAATACTCTTGTAGTCTTGCAACATGGTAACGATGAGTTAGCAACATGCCTTACAAGGAGAGAAAAAGCACCGTGCATGCCGATTGGTGGAAGTAAGGTGGTACGATCGTGCCTTATTAGGAAGGCAACAGACGGGTCTGACATGGATTGGACGAACCACTGAATTGCCGCATTGCAGAGATATTGTATTTAAGTGCCTAGCTCGATACATAAACGGGTCTCTCTGGTTAGACCAGATCTGAGCCTGGGAGCTCTCTGGCTAACTAGGGAACCCACTGCTTAAGCCTCAATAAAGCTTGCCTTGAGTGCTTCAAGTAGTGTGTGCCCGTCTGTTGTGTGACTCTGGTAACTAGAGATCCCTCAGACCCTTTTAGTCAGTGTGGAAAATCTCTAGCAGTGGCGCCCGAACAGGGACTTGAAAGCGAAAGGGAAACCAGAGGAGCTCTCTCGACGCAGGACTCGGCTTGCTGAAGCGCGCACGGCAAGAGGCGAGGGGCGGCGACTGGTGAGTACGCCAAAAATTTTGACTAGCGGAGGCTAGAAGGAGAGAGATGGGTGCGAGAGCGTCAGTATTAAGCGGGGGAGAATTAGATCGCGATGGGAAAAAATTCGGTTAAGGCCAGGGGGAAAGAAAAAATATAAATTAAAACATATAGTATGGGCAAGCAGGGAGCTAGAACGATTCGCAGTTAATCCTGGCCTGTTAGAAACATCAGAAGGCTGTAGACAAATACTGGGACAGCTACAACCATCCCTTCAGACAGGATCAGAAGAACTTAGATCATTATATAATACAGTAGCAACCCTCTATTGTGTGCATCAAAGGATAGAGATAAAAGACACCAAGGAAGCTTTAGACAAGATAGAGGAAGAGCAAAACAAAAGTAAGACCACCGCACAGCAAGCGGCCGCTGATCTTCAGACCTGGAGGAGGAGATATGAGGGACAATTGGAGAAGTGAATTATATAAATATAAAGTAGTAAAAATTGAACCATTAGGAGTAGCACCCACCAAGGCAAAGAGAAGAGTGGTGCAGAGAGAAAAAAGAGCAGTGGGAATAGGAGCTTTGTTCCTTGGGTTCTTGGGAGCAGCAGGAAGCACTATGGGCGCAGCGTCAATGACGCTGACGGTACAGGCCAGACAATTATTGTCTGGTATAGTGCAGCAGCAGAACAATTTGCTGAGGGCTATTGAGGCGCAACAGCATCTGTTGCAACTCACAGTCTGGGGCATCAAGCAGCTCCAGGCAAGAATCCTGGCTGTGGAAAGATACCTAAAGGATCAACAGCTCCTGGGGATTTGGGGTTGCTCTGGAAAACTCATTTGCACCACTGCTGTGCCTTGGAATGCTAGTTGGAGTAATAAATCTCTGGAACAGATTTGGAATCACACGACCTGGATGGAGTGGGACAGAGAAATTAACAATTACACAAGCTTAATACACTCCTTAATTGAAGAATCGCAAAACCAGCAAGAAAAGAATGAACAAGAATTATTGGAATTAGATAAATGGGCAAGTTTGTGGAATTGGTTTAACATAACAAATTGGCTGTGGTATATAAAATTATTCATAATGATAGTAGGAGGCTTGGTAGGTTTAAGAATAGTTTTTGCTGTACTTTCTATAGTGAATAGAGTTAGGCAGGGATATTCACCATTATCGTTTCAGACCCACCTCCCAACCCCGAGGGGACCCGACAGGCCCGAAGGAATAGAAGAAGAAGGTGGAGAGAGAGACAGAGACAGATCCATTCGATTAGTGAACGGATCTCGACGGTATCGATAAGCTTGGGAGTTCCGCGTTACATAACTTACGGTAAATGGCCCGCCTGGCTGACCGCCCAACGACCCCCGCCCATTGACGTCAATAATGACGTATGTTCCCATAGTAACGCCAATAGGGACTTTCCATTGACGTCAATGGGTGGAGTATTTACGGTAAACTGCCCACTTGGCAGTACATCAAGTGTATCATATGCCAAGTACGCCCCCTATTGACGTCAATGACGGTAAATGGCCCGCCTGGCATTATGCCCAGTACATGACCTTATGGGACTTTCCTACTTGGCAGTACATCTACGTATTAGTCATCGCTATTACCATGGTGATGCGGTTTTGGCAGTACATCAATGGGCGTGGATAGCGGTTTGACTCACGGGGATTTCCAAGTCTCCACCCCATTGACGTCAATGGGAGTTTGTTTTGGCACCAAAATCAACGGGACTTTCCAAAATGTCGTAACAACTCCGCCCCATTGACGCAAATGGGCGGTAGGCGTGTACGGTGGGAGGTCTATATAAGCAGAGCTCGTTTAGTGAACCGTCAGATCGCCTGGAGACGCCATCCACGCTGTTTTGACCTCCATAGAAGACACCGACTCTAGAGGATCCACTAGTCCAGTGTGGTGGAATTCTGCAGATATCAACAAGTTTGTACAAAAAAGCTGAACGAGAAACGTAAAATGATATAAATATCAATATATTAAATTAGATTTTGCATAAAAAACAGACTACATAATACTGTAAAACACAACATATCCAGTCACTATGGCGGCCGCATTAGGCACCCCAGGCTTTACACTTTATGCTTCCGGCTCGTATAATGTGTGGATTTTGAGTTAGGATCCGGCGAGATTTTCAGGAGCTAAGGAAGCTAAAATGGAGAAAAAAATCACTGGATATACCACCGTTGATATATCCCAATGGCATCGTAAAGAACATTTTGAGGCATTTCAGTCAGTTGCTCAATGTACCTATAACCAGACCGTTCAGCTGGATATTACGGCCTTTTTAAAGACCGTAAAGAAAAATAAGCACAAGTTTTATCCGGCCTTTATTCACATTCTTGCCCGCCTGATGAATGCTCATCCGGAATTCCGTATGGCAATGAAAGACGGTGAGCTGGTGATATGGGATAGTGTTCACCCTTGTTACACCGTTTTCCATGAGCAAACTGAAACGTTTTCATCGCTCTGGAGTGAATACCACGACGATTTCCGGCAGTTTCTACACATATATTCGCAAGATGTGGCGTGTTACGGTGAAAACCTGGCCTATTTCCCTAAAGGGTTTATTGAGAATATGTTTTTCGTCTCAGCCAATCCCTGGGTGAGTTTCACCAGTTTTGATTTAAACGTGGCCAATATGGACAACTTCTTCGCCCCCGTTTTCACCATGGGCAAATATTATACGCAAGGCGACAAGGTGCTGATGCCGCTGGCGATTCAGGTTCATCATGCCGTCTGTGATGGCTTCCATGTCGGCAGAATGCTTAATGAATTACAACAGTACTGCGATGAGTGGCAGGGCGGGGCGTAAAGATCTGGATCCGGCTTACTAAAAGCCAGATAACAGTATGCGTATTTGCGCGCTGATTTTTGCGGTATAAGAATATATACTGATATGTATACCCGAAGTATGTCAAAAAGAGGTGTGCTATGAAGCAGCGTATTACAGTGACAGTTGACAGCGACAGCTATCAGTTGCTCAAGGCATATATGATGTCAATATCTCCGGTCTGGTAAGCACAACCATGCAGAATGAAGCCCGTCGTCTGCGTGCCGAACGCTGGAAAGCGGAAAATCAGGAAGGGATGGCTGAGGTCGCCCGGTTTATTGAAATGAACGGCTCTTTTGCTGACGAGAACAGGGACTGGTGAAATGCAGTTTAAGGTTTACACCTATAAAAGAGAGAGCCGTTATCGTCTGTTTGTGGATGTACAGAGTGATATTATTGACACGCCCGGGCGACGGATGGTGATCCCCCTGGCCAGTGCACGTCTGCTGTCAGATAAAGTCTCCCGTGAACTTTACCCGGTGGTGCATATCGGGGATGAAAGCTGGCGCATGATGACCACCGATATGGCCAGTGTGCCGGTCTCCGTTATCGGGGAAGAAGTGGCTGATCTCAGCCACCGCGAAAATGACATCAAAAACGCCATTAACCTGATGTTCTGGGGAATATAAATGTCAGGCTCCGTTATACACAGCCAGTCTGCAGGTCGACCATAGTGACTGGATATGTTGTGTTTTACAGTATTATGTAGTCTGTTTTTTATGCAAAATCTAATTTAATATATTGATATTTATATCATTTTACGTTTCTCGTTCAGCTTTCTTGTACAAAGTGGTTGATATCCAGCACAGTGGCGGCCGCTCGAGTCTAGAGGGCCCGCGGTTCGAAGGTAAGCCTATCCCTAACCCTCTCCTCGGTCTCGATTCTACGCGTACCGGTTAGTAATGAGTTTGGAATTAATTCTGTGGAATGTGTGTCAGTTAGGGTGTGGAAAGTCCCCAGGCTCCCCAGGCAGGCAGAAGTATGCAAAGCATGCATCTCAATTAGTCAGCAACCAGGTGTGGAAAGTCCCCAGGCTCCCCAGCAGGCAGAAGTATGCAAAGCATGCATCTCAATTAGTCAGCAACCATAGTCCCGCCCCTAACTCCGCCCATCCCGCCCCTAACTCCGCCCAGTTCCGCCCATTCTCCGCCCCATGGCTGACTAATTTTTTTTATTTATGCAGAGGCCGAGGCCGCCTCTGCCTCTGAGCTATTCCAGAAGTAGTGAGGAGGCTTTTTTGGAGGCCTAGGCTTTTGCAAAAAGCTCCCGGGAGCTTGTATATCCATTTTCGGATCTGATCAGCACGTGTTGACAATTAATCATCGGCATAGTATATCGGCATAGTATAATACGACAAGGTGAGGAACTAAACCATGGCCAAGCCTTTGTCTCAAGAAGAATCCACCCTCATTGAAAGAGCAACGGCTACAATCAACAGCATCCCCATCTCTGAAGACTACAGCGTCGCCAGCGCAGCTCTCTCTAGCGACGGCCGCATCTTCACTGGTGTCAATGTATATCATTTTACTGGGGGACCTTGTGCAGAACTCGTGGTGCTGGGCACTGCTGCTGCTGCGGCAGCTGGCAACCTGACTTGTATCGTCGCGATCGGAAATGAGAACAGGGGCATCTTGAGCCCCTGCGGACGGTGCCGACAGGTGCTTCTCGATCTGCATCCTGGGATCAAAGCCATAGTGAAGGACAGTGATGGACAGCCGACGGCAGTTGGGATTCGTGAATTGCTGCCCTCTGGTTATGTGTGGGAGGGCTAAGCACAATTCGAGCTCGGTACCTTTAAGACCAATGACTTACAAGGCAGCTGTAGATCTTAGCCACTTTTTAAAAGAAAAGGGGGGACTGGAAGGGCTAATTCACTCCCAACGAAGACAAGATCTGCTTTTTGCTTGTACTGGGTCTCTCTGGTTAGACCAGATCTGAGCCTGGGAGCTCTCTGGCTAACTAGGGAACCCACTGCTTAAGCCTCAATAAAGCTTGCCTTGAGTGCTTCAAGTAGTGTGTGCCCGTCTGTTGTGTGACTCTGGTAACTAGAGATCCCTCAGACCCTTTTAGTCAGTGTGGAAAATCTCTAGCAGTAGTAGTTCATGTCATCTTATTATTCAGTATTTATAACTTGCAAAGAAATGAATATCAGAGAGTGAGAGGAACTTGTTTATTGCAGCTTATAATGGTTACAAATAAAGCAATAGCATCACAAATTTCACAAATAAAGCATTTTTTTCACTGCATTCTAGTTGTGGTTTGTCCAAACTCATCAATGTATCTTATCATGTCTGGCTCTAGCTATCCCGCCCCTAACTCCGCCCATCCCGCCCCTAACTCCGCCCAGTTCCGCCCATTCTCCGCCCCATGGCTGACTAATTTTTTTTATTTATGCAGAGGCCGAGGCCGCCTCGGCCTCTGAGCTATTCCAGAAGTAGTGAGGAGGCTTTTTTGGAGGCCTAGGGACGTACCCAATTCGCCCTATAGTGAGTCGTATTACGCGCGCTCACTGGCCGTCGTTTTACAACGTCGTGACTGGGAAAACCCTGGCGTTACCCAACTTAATCGCCTTGCAGCACATCCCCCTTTCGCCAGCTGGCGTAATAGCGAAGAGGCCCGCACCGATCGCCCTTCCCAACAGTTGCGCAGCCTGAATGGCGAATGGGACGCGCCCTGTAGCGGCGCATTAAGCGCGGCGGGTGTGGTGGTTACGCGCAGCGTGACCGCTACACTTGCCAGCGCCCTAGCGCCCGCTCCTTTCGCTTTCTTCCCTTCCTTTCTCGCCACGTTCGCCGGCTTTCCCCGTCAAGCTCTAAATCGGGGGCTCCCTTTAGGGTTCCGATTTAGTGCTTTACGGCACCTCGACCCCAAAAAACTTGATTAGGGTGATGGTTCACGTAGTGGGCCATCGCCCTGATAGACGGTTTTTCGCCCTTTGACGTTGGAGTCCACGTTCTTTAATAGTGGACTCTTGTTCCAAACTGGAACAACACTCAACCCTATCTCGGTCTATTCTTTTGATTTATAAGGGATTTTGCCGATTTCGGCCTATTGGTTAAAAAATGAGCTGATTTAACAAAAATTTAACGCGAATTTTAACAAAATATTAACGCTTACAATTTAGGTGGCACTTTTCGGGGAAATGTGCGCGGAACCCCTATTTGTTTATTTTTCTAAATACATTCAAATATGTATCCGCTCATGAGACAATAACCCTGATAAATGCTTCAATAATATTGAAAAAGGAAGAGTATGAGTATTCAACATTTCCGTGTCGCCCTTATTCCCTTTTTTGCGGCATTTTGCCTTCCTGTTTTTGCTCACCCAGAAACGCTGGTGAAAGTAAAAGATGCTGAAGATCAGTTGGGTGCACGAGTGGGTTACATCGAACTGGATCTCAACAGCGGTAAGATCCTTGAGAGTTTTCGCCCCGAAGAACGTTTTCCAATGATGAGCACTTTTAAAGTTCTGCTATGTGGCGCGGTATTATCCCGTATTGACGCCGGGCAAGAGCAACTCGGTCGCCGCATACACTATTCTCAGAATGACTTGGTTGAGTACTCACCAGTCACAGAAAAGCATCTTACGGATGGCATGACAGTAAGAGAATTATGCAGTGCTGCCATAACCATGAGTGATAACACTGCGGCCAACTTACTTCTGACAACGATCGGAGGACCGAAGGAGCTAACCGCTTTTTTGCACAACATGGGGGATCATGTAACTCGCCTTGATCGTTGGGAACCGGAGCTGAATGAAGCCATACCAAACGACGAGCGTGACACCACGATGCCTGTAGCAATGGCAACAACGTTGCGCAAACTATTAACTGGCGAACTACTTACTCTAGCTTCCCGGCAACAATTAATAGACTGGATGGAGGCGGATAAAGTTGCAGGACCACTTCTGCGCTCGGCCCTTCCGGCTGGCTGGTTTATTGCTGATAAATCTGGAGCCGGTGAGCGTGGGTCTCGCGGTATCATTGCAGCACTGGGGCCAGATGGTAAGCCCTCCCGTATCGTAGTTATCTACACGACGGGGAGTCAGGCAACTATGGATGAACGAAATAGACAGATCGCTGAGATAGGTGCCTCACTGATTAAGCATTGGTAACTGTCAGACCAAGTTTACTCATATATACTTTAGATTGATTTAAAACTTCATTTTTAATTTAAAAGGATCTAGGTGAAGATCCTTTTTGATAATCTCATGACCAAAATCCCTTAACGTGAGTTTTCGTTCCACTGAGCGTCAGACCCCGTAGAAAAGATCAAAGGATCTTCTTGAGATCCTTTTTTTCTGCGCGTAATCTGCTGCTTGCAAACAAAAAAACCACCGCTACCAGCGGTGGTTTGTTTGCCGGATCAAGAGCTACCAACTCTTTTTCCGAAGGTAACTGGCTTCAGCAGAGCGCAGATACCAAATACTGTTCTTCTAGTGTAGCCGTAGTTAGGCCACCACTTCAAGAACTCTGTAGCACCGCCTACATACCTCGCTCTGCTAATCCTGTTACCAGTGGCTGCTGCCAGTGGCGATAAGTCGTGTCTTACCGGGTTGGACTCAAGACGATAGTTACCGGATAAGGCGCAGCGGTCGGGCTGAACGGGGGGTTCGTGCACACAGCCCAGCTTGGAGCGAACGACCTACACCGAACTGAGATACCTACAGCGTGAGCTATGAGAAAGCGCCACGCTTCCCGAAGGGAGAAAGGCGGACAGGTATCCGGTAAGCGGCAGGGTCGGAACAGGAGAGCGCACGAGGGAGCTTCCAGGGGGAAACGCCTGGTATCTTTATAGTCCTGTCGGGTTTCGCCACCTCTGACTTGAGCGTCGATTTTTGTGATGCTCGTCAGGGGGGCGGAGCCTATGGAAAAACGCCAGCAACGCGGCCTTTTTACGGTTCCTGGCCTTTTGCTGGCCTTTTGCTCACATGTTCTTTCCTGCGTTATCCCCTGATTCTGTGGATAACCGTATTACCGCCTTTGAGTGAGCTGATACCGCTCGCCGCAGCCGAACGACCGAGCGCAGCGAGTCAGTGAGCGACGAAGCGGAAGAGCGCCCAATACGCAAACCGCCTCTCCCCGCGCGTTGGCCGATTCATTAATGCAGCTGGCACGACAGGTTTCCCGACTGGAAAGCGGGCAGTGAGCGCAACGCAATTAATGTGAGTTAGCTCACTCATTAGGCACCCCAGGCTTTACACTTTATGCTTCCGGCTCGTATGTTGTGTGGAATTGTGAGCGGATAACAATTTCACACAGGAAACAGCTATGACCATGATTACGCCAAGCGCGCAATTAACCCTCACTAAAGGGAACAAAAGCTGGAGCTGCAAGCTT

[1250] TABLE 18 Nucleotide sequence of plasmid pLenti6/V5-D-TOPO ™.AATGTAGTCTTATGCAATACTCTTGTAGTCTTGCAACATGGTAACGATGAGTTAGCAACATGCCTTACAAGGAGAGAAAAAGCACCGTGCATGCCGATTGGTGGAAGTAAGGTGGTACGATcGTGCCTTATTAGGAAGGCAACAGACGGGTCTGACATGGATTGGACGAACCACTGAATTGCCGCATTGCAGAGATATTGTATTTAAGTGCCTAGCTCGATACATAAACGGGTCTCTCTGGTTAGACCAGATCTGAGCCTGGGAGCTCTCTGGCTAACTAGGGAACCCACTGCTTAAGCCTCAATAAAGCTTGCCTTGAGTGCTTCAAGTAGTGTGTGCCCGTCTGTTGTGTGACTCTGGTAACTAGAGATCCCTCAGACCCTTTTAGTCAGTGTGGAAAATCTCTAGCAGTGGCGCCCGAACAGGGACTTGAAAGCGAAAGGGAAACCAGAGGAGCTCTCTCGACGCAGGACTCGGCTTGCTGAAGCGCGCACGGCAAGAGGCGAGGGGCGGCGACTGGTGAGTACGCCAAAAATTTTGACTAGCGGAGGCTAGAAGGAGAGAGATGGGTGCGAGAGCGTCAGTATTAAGCGGGGGAGAATTAGATCGCGATGGGAAAAAATTCGGTTAAGGCCAGGGGGAAAGAAAAAATATAAATTAAAACATATAGTATGGGCAAGCAGGGAGCTAGAACGATTCGCAGTTAATCCTGGCCTGTTAGAAACATCAGAAGGCTGTAGACAAATACTGGGACAGCTACAACCATCCCTTCAGACAGGATCAGAAGAACTTAGATCATTATATAATACAGTAGCAACCCTCTATTGTGTGCATCAAAGGATAGAGATAAAAGACACCAAGGAAGCTTTAGACAAGATAGAGGAAGAGCAAAACAAAAGTAAGACCACCGCACAGCAAGCGGCCGCTGATCTTCAGACCTGGAGGAGGAGATATGAGGGACAATTGGAGAAGTGAATTATATAAATATAAAGTAGTAAAAATTGAACCATTAGGAGTAGCACCCACCAAGGCAAAGAGAAGAGTGGTGCAGAGAGAAAAAAGAGCAGTGGGAATAGGAGCTTTGTTCCTTGGGTTCTTGGGAGCAGCAGGAAGCACTATGGGCGCAGCGTCAATGACGCTGACGGTACAGGCCAGACAATTATTGTCTGGTATAGTGCAGCAGCAGAACAATTTGCTGAGGGCTATTGAGGCGCAACAGCATCTGTTGCAACTCACAGTCTGGGGCATCAAGCAGCTCCAGGCAAGAATCCTGGCTGTGGAAAGATACCTAAAGGATCAACAGCTCCTGGGGATTTGGGGTTGCTCTGGAAAACTCATTTGCACCACTGCTGTGCCTTGGAATGCTAGTTGGAGTAATAAATCTCTGGAACAGATTTGGAATCACACGACCTGGATGGAGTGGGACAGAGAAATTAACAATTACACAAGCTTAATACACTCCTTAATTGAAGAATCGCAAAACCAGCAAGAAAAGAATGAACAAGAATTATTGGAATTAGATAAATGGGCAAGTTTGTGGAATTGGTTTAACATAACAAATTGGCTGTGGTATATAAAATTATTCATAATGATAGTAGGAGGCTTGGTAGGTTTAAGAATAGTTTTTGCTGTACTTTCTATAGTGAATAGAGTTAGGCAGGGATATTCACCATTATCGTTTCAGACCCACCTCCCAACCCCGAGGGGACCCGACAGGCCCGAAGGAATAGAAGAAGAAGGTGGAGAGAGAGACAGAGACAGATCCATTCGATTAGTGAACGGATCTCGACGGTATCGATAAGCTTGGGAGTTCCGCGTTACATAACTTACGGTAAATGGCCCGCCTGGCTGACCGCCCAACGACCCCCGCCCATTGACGTCAATAATGACGTATGTTCCCATAGTAACGCCAATAGGGACTTTCCATTGACGTCAATGGGTGGAGTATTTACGGTAAACTGCCCACTTGGCAGTACATCAAGTGTATCATATGCCAAGTACGCCCCCTATTGACGTCAATGACGGTAAATGGCCCGCCTGGCATTATGCCCAGTACATGACCTTATGGGACTTTCCTACTTGGCAGTACATCTACGTATTAGTCATCGCTATTACCATGGTGATGCGGTTTTGGCAGTACATCAATGGGCGTGGATAGCGGTTTGACTCACGGGGATTTCCAAGTCTCCACCCCATTGACGTCAATGGGAGTTTGTTTTGGCACCAPAATCAACGGGACTTTCCAAAATGTCGTAACAACTCCGCCCCATTGACGCAAATGGGCGGTAGGCGTGTACGGTGGGAGGTCTATATAAGCAGAGCTCGTTTAGTGAACCGTCAGATCGCCTGGAGACGCCATCCACGCTGTTTTGACCTCCATAGAAGACACCGACTCTAGAGGATCCACTAGTCCAGTGTGGTGGAATTGATCCCTTCACCAAGGGCTCGAGTCTAGAGGGCCCGCGGTTCGAAGGTAAGCCTATCCCTAACCCTCTCCTCGGTCTCGATTCTACGCGTACCGGTTAGTAATGAGTTTGGAATTAATTCTGTGGAATGTGTGTCAGTTAGGGTGTGGAAAGTCCCCAGGCTCCCCAGGCAGGCAGAAGTATGCAAAGCATGCATCTCAATTAGTCAGCAACCAGGTGTGGAAAGTCCCCAGGCTCCCCAGCAGGCAGAAGTATGCAAAGCATGCATCTCAATTAGTCAGCAACCATAGTCCCGCCCCTAACTCCGCCCATCCCGCCCCTAACTCCGCCCAGTTCCGCCCATTCTCCGCCCCATGGCTGACTAATTTTTTTTATTTATGCAGAGGCCGAGGCCGCCTCTGCCTCTGAGCTATTCCAGAAGTAGTGAGGAGGCTTTTTTGGAGGCCTAGGCTTTTGCAAAAAGCTCCCGGGAGCTTGTATATCCATTTTCGGATCTGATCAGCACGTGTTGACAATTAATCATCGGCATAGTATATCGGCATAGTATAATACGACAAGGTGAGGAACTAAACCATGGCCAAGCCTTTGTCTCAAGAAGAATCCACCCTCATTGAAAGAGCAACGGCTACAATCAACAGCATCCCCATCTCTGAAGACTACAGCGTCGCCAGCGCAGCTCTCTCTAGCGACGGCCGCATCTTCACTGGTGTCAATGTATATCATTTTACTGGGGGACCTTGTGCAGAACTCGTGGTGCTGGGCACTGCTGCTGCTGCGGCAGCTGGCAACCTGACTTGTATCGTCGCGATCGGAAATGAGAACAGGGGCATCTTGAGCCCCTGCGGACGGTGCCGACAGGTGCTTCTCGATCTGCATCCTGGGATCAAAGCCATAGTGAAGGACAGTGATGGACAGCCGACGGCAGTTGGGATTCGTGAATTGCTGCCCTCTGGTTATGTGTGGGAGGGCTAAGCACAATTCGAGCTCGGTACCTTTAAGACCAATGACTTACAAGGCAGCTGTAGATCTTAGCCACTTTTTAAAAGAAAAGGGGGGACTGGAAGGGCTAATTCACTCCCAACGAAGACAAGATCTGCTTTTTGCTTGTACTGGGTCTCTCTGGTTAGACCAGATCTGAGCCTGGGAGCTCTCTGGCTAACTAGGGAACCCACTGCTTAAGCCTCAATAAAGCTTGCCTTGAGTGCTTCAAGTAGTGTGTGCCCGTCTGTTGTGTGACTCTGGTAACTAGAGATCCCTCAGACCCTTTTAGTCAGTGTGGAAAATCTCTAGCAGTAGTAGTTCATGTCATCTTATTATTCAGTATTTATAACTTGCAAAGAAATGAATATCAGAGAGTGAGAGGAACTTGTTTATTGCAGCTTATAATGGTTACAAATAAAGCAATAGCATCACAAATTTCACAAATAAAGCATTTTTTTCACTGCATTCTAGTTGTGGTTTGTCCAAACTCATCAATGTATCTTATCATGTCTGGCTCTAGCTATCCCGCCCCTAACTCCGCCCATCCCGCCCCTAACTCCGCCCAGTTCCGCCCATTCTCCGCCCCATGGCTGACTAATTTTTTTTATTTATGCAGAGGCCGAGGCCGCCTCGGCCTCTGAGCTATTCCAGAAGTAGTGAGGAGGCTTTTTTGGAGGCCTAGGGACGTACCCAATTCGCCCTATAGTGAGTCGTATTACGCGCGCTCACTGGCCGTCGTTTTACAACGTCGTGACTGGGAAAACCCTGGCGTTACCCAACTTAATCGCCTTGCAGCACATCCCCCTTTCGCCAGCTGGCGTAATAGCGAAGAGGCCCGCACCGATCGCCCTTCCCAACAGTTGCGCAGCCTGAATGGCGAATGGGACGCGCCCTGTAGCGGCGCATTAAGCGCGGCGGGTGTGGTGGTTACGCGCAGCGTGACCGCTACACTTGCCAGCGCCCTAGCGCCCGCTCCTTTCGCTTTCTTCCCTTCCTTTCTCGCCACGTTCGCCGGCTTTCCCCGTCAAGCTCTAAATCGGGGGCTCCCTTTAGGGTTCCGATTTAGTGCTTTACGGCACCTCGACCCCAAAAPACTTGATTAGGGTGATGGTTCACGTAGTGGGCCATCGCCCTGATAGACGGTTTTTCGCCCTTTGACGTTGGAGTCCACGTTCTTTAATAGTGGACTCTTGTTCCAAACTGGAACAACACTCAACCCTATCTCGGTCTATTCTTTTGATTTATAAGGGATTTTGCCGATTTCGGCCTATTGGTTAAAAAATGAGCTGATTTAACAAAAATTTAACGCGAATTTTAACAAAATATTAACGCTTACAATTTAGGTGGCACTTTTCGGGGAAATGTGCGCGGAACCCCTATTTGTTTATTTTTCTAAATACATTCAAATATGTATCCGCTCATGAGACAATAACCCTGATAAATGCTTCAATAATATTGAAAAAGGAAGAGTATGAGTATTCAACATTTCCGTGTCGCCCTTATTCCCTTTTTTGCGGCATTTTGCCTTCCTGTTTTTGCTCACCCAGAAACGCTGGTGAAAGTAAAAGATGCTGAAGATCAGTTGGGTGCACGAGTGGGTTACATCGAACTGGATCTCAACAGCGGTAAGATCCTTGAGAGTTTTCGCCCCGAAGAACGTTTTCCAATGATGAGCACTTTTAAAGTTCTGCTATGTGGCGCGGTATTATCCCGTATTGACGCCGGGCAAGAGCAACTCGGTCGCCGCATACACTATTCTCAGAATGACTTGGTTGAGTACTCACCAGTCACAGAAAAGCATCTTACGGATGGCATGACAGTAAGAGAATTATGCAGTGCTGCCATAACCATGAGTGATAACACTGCGGCCAACTTACTTCTGACAACGATCGGAGGACCGAAGGAGCTAACCGCTTTTTTGCACAACATGGGGGATCATGTAACTCGCCTTGATCGTTGGGAACCGGAGCTGAATGAAGCCATACCAAACGACGAGCGTGACACCACGATGCCTGTAGCAATGGCAACAACGTTGCGCAAACTATTAACTGGCGAACTACTTACTCTAGCTTCCCGGCAACAATTAATAGACTGGATGGAGGCGGATAAAGTTGCAGGACCACTTCTGCGCTCGGCCCTTCCGGCTGGCTGGTTTATTGCTGATAAATCTGGAGCCGGTGAGCGTGGGTCTCGCGGTATCATTGCAGCACTGGGGCCAGATGGTAAGCCCTCCCGTATCGTAGTTATCTACACGACGGGGAGTCAGGCAACTATGGATGAACGAAATAGACAGATCGCTGAGATAGGTGCCTCACTGATTAAGCATTGGTAACTGTCAGACCAAGTTTACTCATATATACTTTAGATTGATTTAAAACTTCATTTTTAATTTAAAAGGATCTAGGTGAAGATCCTTTTTGATAATCTCATGACCAAAATCCCTTAACGTGAGTTTTCGTTCCACTGAGCGTCAGACCCCGTAGAAAAGATCAAAGGATCTTCTTGAGATCCTTTTTTTCTGCGCGTAATCTGCTGCTTGCAAACAAAAAAACCACCGCTACCAGCGGTGGTTTGTTTGCCGGATCAAGAGCTACCAACTCTTTTTCCGAAGGTAACTGGCTTCAGCAGAGCGCAGATACCAAATACTGTTCTTCTAGTGTAGCCGTAGTTAGGCCACCACTTCAAGAACTCTGTAGCACCGCCTACATACCTCGCTCTGCTAATCCTGTTACCAGTGGCTGCTGCCAGTGGCGATAAGTCGTGTCTTACCGGGTTGGACTCAAGACGATAGTTACCGGATAAGGCGCAGCGGTCGGGCTGAACGGGGGGTTCGTGCACACAGCCCAGCTTGGAGCGAACGACCTACACCGAACTGAGATACCTACAGCGTGAGCTATGAGAAAGCGCCACGCTTCCCGAAGGGAGAAAGGCGGACAGGTATCCGGTAAGCGGCAGGGTCGGAACAGGAGAGCGCACGAGGGAGCTTCCAGGGGGAAACGCCTGGTATCTTTATAGTCCTGTCGGGTTTCGCCACCTCTGACTTGAGCGTCGATTTTTGTGATGCTCGTCAGGGGGGCGGAGCCTATGGAAAAACGCCAGCAACGCGGCCTTTTTACGGTTCCTGGCCTTTTGCTGGCCTTTTGCTCACATGTTCTTTCCTGCGTTATCCCCTGATTCTGTGGATAACCGTATTACCGCCTTTGAGTGAGCTGATACCGCTCGCCGCAGCCGAACGACCGAGCGCAGCGAGTCAGTGAGCGAGGAAGCGGAAGAGCGCCCAATACGCAAACCGCCTCTCCCCGCGCGTTGGCCGATTCATTAATGCAGCTGGCACGACAGGTTTCCCGACTGGAAAGCGGGCAGTGAGCGCAACGCAATTAATGTGAGTTAGCTCACTCATTAGGCACCCCAGGCTTTACACTTTATGCTTCCGGCTCGTATGTTGTGTGGAATTGTGAGCGGATAACAATTTCACACAGGAAACAGCTATGACCATGATTACGCCAAGCGCGCAATTAACCCTCACTAAAGGGAACAAAAGCTGGAGCTGCAAGCTT

[1251] TABLE 19 Nucleotide sequence of pLenti4/V5-DEST.AATGTAGTCTTATGCAATACTCTTGTAGTCTTGCAACATGGTAACGATGAGTTAGCAACATGCCTTACAAGGAGAGAAAAAGCACCGTGCATGCCGATTGGTGGAAGTAAGGTGGTACGATCGTGCCTTATTAGGAAGGCAACAGACGGGTCTGACATGGATTGGACGAACCACTGAATTGCCGCATTGCAGAGATATTGTATTTAAGTGCCTAGCTCGATACATAAACGGGTCTCTCTGGTTAGACCAGATCTGAGCCTGGGAGCTCTCTGGCTAACTAGGGAACCCACTGCTTAAGCCTCAATAAAGCTTGCCTTGAGTGCTTCAAGTAGTGTGTGCCCGTCTGTTGTGTGACTCTGGTAACTAGAGATCCCTCAGACCCTTTTAGTCAGTGTGGAAAATCTCTAGCAGTGGCGCCCGAACAGGGACTTGAAAGCGAAAGGGAAACCAGAGGAGCTCTCTCGACGCAGGACTCGGCTTGCTGAAGCGCGCACGGCAAGAGGCGAGGGGCGGCGACTGGTGAGTACGCCAAAAATTTTGACTAGCGGAGGCTAGAAGGAGAGAGATGGGTGCGAGAGCGTCAGTATTAAGCGGGGGAGAATTAGATCGCGATGGGAAAAAATTCGGTTAAGGCCAGGGGGAAAGAAAAAATATAAATTAAAACATATAGTATGGGCAAGCAGGGAGCTAGAACGATTCGCAGTTAATCCTGGCCTGTTAGAAACATCAGAAGGCTGTAGACAAATACTGGGACAGCTACAACCATCCCTTCAGACAGGATCAGAAGAACTTAGATCATTATATAATACAGTAGCAACCCTCTATTGTGTGCATCAAAGGATAGAGATAAAAGACACCAAGGAAGCTTTAGACAAGATAGAGGAAGAGCAAAACAAAAGTAAGACCACCGCACAGCAAGCGGCCGCTGATCTTCAGACCTGGAGGAGGAGATATGAGGGACAATTGGAGAAGTGAATTATATAAATATAAAGTAGTAAAAATTGAACCATTAGGAGTAGCACCCACCAAGGCAAAGAGAAGAGTGGTGCAGAGAGAAAAAAGAGCAGTGGGAATAGGAGCTTTGTTCCTTGGGTTCTTGGGAGCAGCAGGAAGCACTATGGGCGCAGCGTCAATGACGCTGACGGTACAGGCCAGACAATTATTGTCTGGTATAGTGCAGCAGCAGAACAATTTGCTGAGGGCTATTGAGGCGCAACAGCATCTGTTGCAACTCACAGTCTGGGGCATCPAGCAGCTCCAGGCAAGAATCCTGGCTGTGGAAAGATACCTAAAGGATCAACAGCTCCTGGGGATTTGGGGTTGCTCTGGAAAACTCATTTGCACCACTGCTGTGCCTTGGAATGCTAGTTGGAGTAATAAATCTCTGGAACAGATTTGGAATCACACGACCTGGATGGAGTGGGACAGAGAAATTAACAATTACACAAGCTTAATACACTCCTTAATTGAAGAATCGCAAAACCAGCAAGAAAAGAATGAACAAGAATTATTGGAATTAGATAAATGGGCAAGTTTGTGGAATTGGTTTAACATAACAAATTGGCTGTGGTATATAAAATTATTCATAATGATAGTAGGAGGCTTGGTAGGTTTAAGAATAGTTTTTGCTGTACTTTCTATAGTGAATAGAGTTAGGCAGGGATATTCACCATTATCGTTTCAGACCCACCTCCCAACCCCGAGGGGACCCGACAGGCCCGAAGGAATAGAAGAAGAAGGTGGAGAGAGAGACAGAGACAGATCCATTCGATTAGTGAACGGATCTCGACGGTATCGATAAGCTTGGGAGTTCCGCGTTACATAACTTACGGTAAATGGCCCGCCTGGCTGACCGCCCAACGACCCCCGCCCATTGACGTCAATAATGACGTATGTTCCCATAGTAACGCCAATAGGGACTTTCCATTGACGTCAATGGGTGGAGTATTTACGGTAAACTGCCCACTTGGCAGTACATCAAGTGTATCATATGCCAAGTACGCCCCCTATTGACGTCAATGACGGTAAATGGCCCGCCTGGCATTATGCCCAGTACATGACCTTATGGGACTTTCCTACTTGGCAGTACATCTACGTATTAGTCATCGCTATTACCATGGTGATGCGGTTTTGGCAGTACATCAATGGGCGTGGATAGCGGTTTGACTCACGGGGATTTCCAAGTCTCCACCCCATTGACGTCAATGGGAGTTTGTTTTGGCACCAAAATCAACGGGACTTTCCAAAATGTCGTAACAACTCCGCCCCATTGACGCAAATGGGCGGTAGGCGTGTACGGTGGGAGGTCTATATAAGCAGAGCTCGTTTAGTGAACCGTCAGATCGCCTGGAGACGCCATCCACGCTGTTTTGACCTCCATAGAAGACACCGACTCTAGAGGATCCACTAGTCCAGTGTGGTGGAATTCTGCAGATATCAACAAGTTTGTACAAAAAAGCTGAACGAGAAACGTAAAATGATATAAATATCAATATATTAAATTAGATTTTGCATAAAAAACAGACTACATAATACTGTAAAACACAACATATCCAGTCACTATGGCGGCCGCATTAGGCACCCCAGGCTTTACACTTTATGCTTCCGGCTCGTATAATGTGTGGATTTTGAGTTAGGATCCGGCGAGATTTTCAGGAGCTAAGGAAGCTAAAATGGAGAAAAAAATCACTGGATATACCACCGTTGATATATCCCAATGGCATCGTAAAGAACATTTTGAGGCATTTCAGTCAGTTGCTCAATGTACCTATAACCAGACCGTTCAGCTGGATATTACGGCCTTTTTAAAGACCGTAAAGAAAAATAAGCACAAGTTTTATCCGGCCTTTATTCACATTCTTGCCCGCCTGATGAATGCTCATCCGGAATTCCGTATGGCAATGAAAGACGGTGAGCTGGTGATATGGGATAGTGTTCACCCTTGTTACACCGTTTTCCATGAGCAAACTGAAACGTTTTCATCGCTCTGGAGTGAATACCACGACGATTTCCGGCAGTTTCTACACATATATTCGCAAGATGTGGCGTGTTACGGTGAAAACCTGGCCTATTTCCCTAAAGGGTTTATTGAGAATATGTTTTTCGTCTCAGCCAATCCCTGGGTGAGTTTCACCAGTTTTGATTTAAACGTGGCCAATATGGACAACTTCTTCGCCCCCGTTTTCACCATGGGCAAATATTATACGCAAGGCGACAAGGTGCTGATGCCGCTGGCGATTCAGGTTCATCATGCCGTCTGTGATGGCTTCCATGTCGGCAGAATGCTTAATGAATTACAACAGTACTGCGATGAGTGGCAGGGCGGGGCGTAAAGATCTGGATCCGGCTTACTAAAAGCCAGATAACAGTATGCGTATTTGCGCGCTGATTTTTGCGGTATAAGAATATATACTGATATGTATACCCGAAGTATGTCAAAAAGAGGTGTGCTATGAAGCAGCGTATTACAGTGACAGTTGACAGCGACAGCTATCAGTTGCTCAAGGCATATATGATGTCAATATCTCCGGTCTGGTAAGCACAACCATGCAGAATGAAGCCCGTCGTCTGCGTGCCGAACGCTGGAAAGCGGAAAATCAGGAAGGGATGGCTGAGGTCGCCCGGTTTATTGAAATGAACGGCTCTTTTGCTGACGAGAACAGGGACTGGTGAAATGCAGTTTAAGGTTTACACCTATAAAAGAGAGAGCCGTTATCGTCTGTTTGTGGATGTACAGAGTGATATTATTGACACGCCCGGGCGACGGATGGTGATCCCCCTGGCCAGTGCACGTCTGCTGTCAGATAAAGTCTCCCGTGAACTTTACCCGGTGGTGCATATCGGGGATGAAAGCTGGCGCATGATGACCACCGATATGGCCAGTGTGCCGGTCTCCGTTATCGGGGAAGAAGTGGCTGATCTCAGCCACCGCGAAAATGACATCAAAAACGCCATTAACCTGATGTTCTGGGGAATATAAATGTCAGGCTCCGTTATACACAGCCAGTCTGCAGGTCGACCATAGTGACTGGATATGTTGTGTTTTACAGTATTATGTAGTCTGTTTTTTATGCAAAATCTAATTTAATATATTGATATTTATATCATTTTACGTTTCTCGTTCAGCTTTCTTGTACAAAGTGGTTGATATCCAGCACAGTGGCGGCCGCTCGAGTCTAGAGGGCCCGCGGTTCGAAGGTAAGCCTATCCCTAACCCTCTCCTCGGTCTCGATTCTACGCGTACCGGTTAGTAATGAGTTTGGAATTAATTCTGTGGAATGTGTGTCAGTTAGGGTGTGGAAAGTCCCCAGGCTCCCCAGGCAGGCAGAAGTATGCAAAGCATGCATCTCAATTAGTCAGCAACCAGGTGTGGAAAGTCCCCAGGCTCCCCAGCAGGCAGAAGTATGCAAAGCATGCATCTCAATTAGTCAGCAACCATAGTCCCGCCCCTAACTCCGCCCATCCCGCCCCTAACTCCGCCCAGTTCCGCCCATTCTCCGCCCCATGGCTGACTAATTTTTTTTATTTATGCAGAGGCCGAGGCCGCCTCTGCCTCTGAGCTATTCCAGAAGTAGTGAGGAGGCTTTTTTGGAGGCCTAGGCTTTTGCAAAAAGCTCCCCCTGTTGACAATTAATCATCGGCATAGTATATCGGCATAGTATAATACGACAAGGTGAGGAACTAAACCATGGCCAAGTTGACCAGTGCCGTTCCGGTGCTCACCGCGCGCGACGTCGCCGGAGCGGTCGAGTTCTGGACCGACCGGCTCGGGTTCTCCCGGGACTTCGTGGAGGACGACTTCGCCGGTGTGGTCCGGGACGACGTGACCCTGTTCATCAGCGCGGTCCAGGACCAGGTGGTGCCGGACAACACCCTGGCCTGGGTGTGGGTGCGCGGCCTGGACGAGCTGTACGCCGAGTGGTCGGAGGTCGTGTCCACGAACTTCCGGGACGCCTCCGGGCCGGCCATGACCGAGATCGGCGAGCAGCCGTGGGGGCGGGAGTTCGCCCTGCGCGACCCGGCCGGCAACTGCGTGCACTTCGTGGCCGAGGAGCAGGACTGACACGTGCTACGAGATTTAAATGGTACCTTTAAGACCAATGACTTACAAGGCAGCTGTAGATCTTAGCCACTTTTTAAAAGAAAAGGGGGGACTGGAAGGGCTAATTCACTCCCAACGAAGACAAGATCTGCTTTTTGCTTGTACTGGGTCTCTCTGGTTAGACCAGATCTGAGCCTGGGAGCTCTCTGGCTAACTAGGGAACCCACTGCTTAAGCCTCAATAAAGCTTGCCTTGAGTGCTTCAAGTAGTGTGTGCCCGTCTGTTGTGTGACTCTGGTAACTAGAGATCCCTCAGACCCTTTTAGTCAGTGTGGAAAATCTCTAGCAGTAGTAGTTCATGTCATCTTATTATTCAGTATTTATAACTTGCAAAGAAATGAATATCAGAGAGTGAGAGGAACTTGTTTATTGCAGCTTATAATGGTTACAAATAAAGCAATAGCATCACAAATTTCACAAATAAAGCATTTTTTTCACTGCATTCTAGTTGTGGTTTGTCCAAACTCATCAATGTATCTTATCATGTCTGGCTCTAGCTATCCCGCCCCTAACTCCGCCCATCCCGCCCCTAACTCCGCCCAGTTCCGCCCATTCTCCGCCCCATGGCTGACTAATTTTTTTTATTTATGCAGAGGCCGAGGCCGCCTCGGCCTCTGAGCTATTCCAGAAGTAGTGAGGAGGCTTTTTTGGAGGCCTAGGGACGTACCCAATTCGCCCTATAGTGAGTCGTATTACGCGCGCTCACTGGCCGTCGTTTTACAACGTCGTGACTGGGAAAACCCTGGCGTTACCCAACTTAATCGCCTTGCAGCACATCCCCCTTTCGCCAGCTGGCGTAATAGCGAAGAGGCCCGCACCGATCGCCCTTCCCAACAGTTGCGCAGCCTGAATGGCGAATGGGACGCGCCCTGTAGCGGCGCATTAAGCGCGGCGGGTGTGGTGGTTACGCGCAGCGTGACCGCTACACTTGCCAGCGCCCTAGCGCCCGCTCCTTTCGCTTTCTTCCCTTCCTTTCTCGCCACGTTCGCCGGCTTTCCCCGTCAAGCTCTAAATCGGGGGCTCCCTTTAGGGTTCCGATTTAGTGCTTTACGGCACCTCGACCCCAAAAAACTTGATTAGGGTGATGGTTCACGTAGTGGGCCATCGCCCTGATAGACGGTTTTTCGCCCTTTGACGTTGGAGTCCACGTTCTTTAATAGTGGACTCTTGTTCCAAACTGGAACAACACTCAACCCTATCTCGGTCTATTCTTTTGATTTATAAGGGATTTTGCCGATTTCGGCCTATTGGTTAAAAAATGAGCTGATTTAACAAAAATTTAACGCGAATTTTAACAAAATATTAACGCTTACAATTTAGGTGGCACTTTTCGGGGAAATGTGCGCGGAACCCCTATTTGTTTATTTTTCTAAATACATTCAAATATGTATCCGCTCATGAGACAATAACCCTGATAAATGCTTCAATAATATTGAAAAAGGAAGAGTATGAGTATTCAACATTTCCGTGTCGCCCTTATTCCCTTTTTTGCGGCATTTTGCCTTCCTGTTTTTGCTCACCCAGAAACGCTGGTGAAAGTAAAAGATGCTGAAGATCAGTTGGGTGCACGAGTGGGTTACATCGAACTGGATCTCAACAGCGGTAAGATCCTTGAGAGTTTTCGCCCCGAAGAACGTTTTCCAATGATGAGCACTTTTAAAGTTCTGCTATGTGGCGCGGTATTATCCCGTATTGACGCCGGGCAAGAGCAACTCGGTCGCCGCATACACTATTCTCAGAATGACTTGGTTGAGTACTCACCAGTCACAGAAAAGCATCTTACGGATGGCATGACAGTAAGAGAATTATGCAGTGCTGCCATAACCATGAGTGATAACACTGCGGCCAACTTACTTCTGACAACGATCGGAGGACCGAAGGAGCTAACCGCTTTTTTGCACAACATGGGGGATCATGTAACTCGCCTTGATCGTTGGGAACCGGAGCTGAATGAAGCCATACCAAACGACGAGCGTGACACCACGATGCCTGTAGCAATGGCAACAACGTTGCGCAAACTATTAACTGGCGAACTACTTACTCTAGCTTCCCGGCAACAATTAATAGACTGGATGGAGGCGGATAAAGTTGCAGGACCACTTCTGCGCTCGGCCCTTCCGGCTGGCTGGTTTATTGCTGATAAATCTGGAGCCGGTGAGCGTGGGTCTCGCGGTATCATTGCAGCACTGGGGCCAGATGGTAAGCCCTCCCGTATCGTAGTTATCTACACGACGGGGAGTCAGGCAACTATGGATGAACGAAATAGACAGATCGCTGAGATAGGTGCCTCACTGATTAAGCATTGGTAACTGTCAGACCAAGTTTACTCATATATACTTTAGATTGATTTAAAACTTCATTTTTAATTTAAAAGGATCTAGGTGAAGATCCTTTTTGATAATCTCATGACCAAAATCCCTTAACGTGAGTTTTCGTTCCACTGAGCGTCAGACCCCGTAGAAAAGATCAAAGGATCTTCTTGAGATCCTTTTTTTCTGCGCGTAATCTGCTGCTTGCAAACAAAAAAACCACCGCTACCAGCGGTGGTTTGTTTGCCGGATCAAGAGCTACCAACTCTTTTTCCGAAGGTAACTGGCTTCAGCAGAGCGCAGATACCAAATACTGTTCTTCTAGTGTAGCCGTAGTTAGGCCACCACTTCAAGAACTCTGTAGCACCGCCTACATACCTCGCTCTGCTAATCCTGTTACCAGTGGCTGCTGCCAGTGGCGATAAGTCGTGTCTTACCGGGTTGGACTCAAGACGATAGTTACCGGATAAGGCGCAGCGGTCGGGCTGAACGGGGGGTTCGTGCACACAGCCCAGCTTGGAGCGAACGACCTACACCGAACTGAGATACCTACAGCGTGAGCTATGAGAAAGCGCCACGCTTCCCGAAGGGAGAAAGGCGGACAGGTATCCGGTAAGCGGCAGGGTCGGAACAGGAGAGCGCACGAGGGAGCTTCCAGGGGGAAACGCCTGGTATCTTTATAGTCCTGTCGGGTTTCGCCACCTCTGACTTGAGCGTCGATTTTTGTGATGCTCGTCAGGGGGGCGGAGCCTATGGAAAAACGCCAGCAACGCGGCCTTTTTACGGTTCCTGGCCTTTTGCTGGCCTTTTGCTCACATGTTCTTTCCTGCGTTATCCCCTGATTCTGTGGATAACCGTATTACCGCCTTTGAGTGAGCTGATACCGCTCGCCGCAGCCGAACGACCGAGCGCAGCGAGTCAGTGAGCGAGGAAGCGGAAGAGCGCCCAATACGCAAACCGCCTCTCCCCGCGCGTTGGCCGATTCATTAATGCAGCTGGCACGACAGGTTTCCCGACTGGAAAGCGGGCAGTGAGCGCAACGCAATTAATGTGAGTTAGCTCACTCATTAGGCACCCCAGGCTTTACACTTTATGCTTCCGGCTCGTATGTTGTGTGGAATTGTGAGCGGATAACAATTTCACACAGGAAACAGCTATGACCATGATTACGCCAAGCGCGCAATTAACCCTCACTAAAGGGAACAAAAGCTGGAGCTGCAAGCTT

[1252] TABLE 20 Nucleotide sequence of pLenti6/UbC/V5-DEST.AATGTAGTCTTATGCAATACTCTTGTAGTCTTGCAACATGGTAACGATGAGTTAGCAACATGCCTTACAAGGAGAGAAAAAGCACCGTGCATGCCGATTGGTGGAAGTAAGGTGGTACGATCGTGCCTTATTAGGAAGGCAACAGACGGGTCTGACATGGATTGGACGAACCACTGAATTGCCGCATTGCAGAGATATTGTATTTAAGTGCCTAGCTCGATACATAAACGGGTCTCTCTGGTTAGACCAGATCTGAGCCTGGGAGCTCTCTGGCTAACTAGGGAACCCACTGCTTAAGCCTCAATAAAGCTTGCCTTGAGTGCTTCAAGTAGTGTGTGCCCGTCTGTTGTGTGACTCTGGTAACTAGAGATCCCTCAGACCCTTTTAGTCAGTGTGGAAAATCTCTAGCAGTGGCGCCCGAACAGGGACTTGAAAGCGAAAGGGAAACCAGAGGAGCTCTCTCGACGCAGGACTCGGCTTGCTGAAGCGCGCACGGCAAGAGGCGAGGGGCGGCGACTGGTGAGTACGCCAAAAATTTTGACTAGCGGAGGCTAGAAGGAGAGAGATGGGTGCGAGAGCGTCAGTATTAAGCGGGGGAGAATTAGATCGCGATGGGAAAAAATTCGGTTAAGGCCAGGGGGAAAGAAAAAATATAAATTAAAACATATAGTATGGGCAAGCAGGGAGCTAGAACGATTCGCAGTTAATCCTGGCCTGTTAGAAACATCAGAAGGCTGTAGACAAATACTGGGACAGCTACAACCATCCCTTCAGACAGGATCAGAAGAACTTAGATCATTATATAATACAGTAGCAACCCTCTATTGTGTGCATCAAAGGATAGAGATAAAAGACACCAAGGAAGCTTTAGACAAGATAGAGGAAGAGCAAAACAAAAGTAAGACCACCGCACAGCAAGCGGCCGCTGATCTTCAGACCTGGAGGAGGAGATATGAGGGACAATTGGAGAAGTGAATTATATAAATATAAAGTAGTAAAAATTGAACCATTAGGAGTAGCACCCACCAAGGCAAAGAGAAGAGTGGTGCAGAGAGAAAAAAGAGCAGTGGGAATAGGAGCTTTGTTCCTTGGGTTCTTGGGAGCAGCAGGAAGCACTATGGGCGCAGCGTCAATGACGCTGACGGTACAGGCCAGACAATTATTGTCTGGTATAGTGCAGCAGCAGAACAATTTGCTGAGGGCTATTGAGGCGCAACAGCATCTGTTGCAACTCACAGTCTGGGGCATCAAGCAGCTCCAGGCAAGAATCCTGGCTGTGGAAAGATACCTAAAGGATCAACAGCTCCTGGGGATTTGGGGTTGCTCTGGAAAACTCATTTGCACCACTGCTGTGCCTTGGAATGCTAGTTGGAGTAATAAATCTCTGGAACAGATTTGGAATCACACGACCTGGATGGAGTGGGACAGAGAAATTAACAATTACACAAGCTTAATACACTCCTTAATTGAAGAATCGCAAAACCAGCAAGAAAAGAATGAACAAGAATTATTGGAATTAGATAAATGGGCAAGTTTGTGGAATTGGTTTAACATAACAAATTGGCTGTGGTATATAAAATTATTCATAATGATAGTAGGAGGCTTGGTAGGTTTAAGAATAGTTTTTGCTGTACTTTCTATAGTGAATAGAGTTAGGCAGGGATATTCACCATTATCGTTTCAGACCCACCTCCCAACCCCGAGGGGACCCGACAGGCCCGAAGGAATAGAAGAAGAAGGTGGAGAGAGAGACAGAGACAGATCCATTCGATTAGTGAACGGATCTCGACGGTATCGGATCTGGCCTCCGCGCCGGGTTTTGGCGCCTCCCGCGGGCGCCCCCCTCCTCACGGCGAGCGCTGCCACGTCAGACGAAGGGCGCAGGAGCGTCCTGATCCTTCCGCCCGGACGCTCAGGACAGCGGCCCGCTGCTCATAAGACTCGGCCTTAGAACCCCAGTATCAGCAGAAGGACATTTTAGGACGGGACTTGGGTGACTCTAGGGCACTCGTTTTCTTTCCAGAGAGCGGAACAGGCGAGGAAAAGTAGTCCCTTCTCGGCGATTCTGCGGAGGGATCTCCGTGGGGCGGTGAACGCCGATGATTATATAAGGACGCGCCGGGTGTGGCACAGCTAGTTCCGTCGCAGCCGGGATTTGGGTCGCGGTTCTTGTTTGTGGATCGCTGTGATCGTCACTTGGTGAGTAGCGGGCTGCTGGGCTGGCCGGGGCTTTCGTGGCCGCCGGGCCGCTCGGTGGGACGGAAGCGTGTGGAGAGACCGCCAAGGGCTGTAGTCTGGGTCCGCGAGCAAGGTTGCCCTGAACTGGGGGTTGGGGGGAGCGCAGCAAAATGGCGGCTGTTCCCGAGTCTTGAATGGAAGACGCTTGTGAGGCGGGCTGTGAGGTCGTTGAAACAAGGTGGGGGGCATGGTGGGCGGCAAGAACCCAAGGTCTTGAGGCCTTCGCTAATGCGGGAAAGCTCTTATTCGGGTGAGATGGGCTGGGGCACCATCTGGGGACCCTGACGTGAAGTTTGTCACTGACTGGAGAACTCGGTTTGTCGTCTGTTGCGGGGGCGGCAGTTATGCGGTGCCGTTGGGCAGTGCACCCGTACCTTTGGGAGCGCGCGCCCTCGTCGTGTCGTGACGTCACCCGTTCTGTTGGCTTATAATGCAGGGTGGGGCCACCTGCCGGTAGGTGTGCGGTAGGCTTTTCTCCGTCGCAGGACGCAGGGTTCGGGCCTAGGGTAGGCTCTCCTGAATCGACAGGCGCCGGACCTCTGGTGAGGGGAGGGATAAGTGAGGCGTCAGTTTCTTTGGTCGGTTTTATGTACCTATCTTCTTAAGTAGCTGAAGCTCCGGTTTTGAACTATGCGCTCGGGGTTGGCGAGTGTGTTTTGTGAAGTTTTTTAGGCACCTTTTGAAATGTAATCATTTGGGTCAATATGTAATTTTCAGTGTTAGACTAGTAAATTGTCCGCTAAATTCTGGCCGTTTTTGGCTTTTTTGTTAGACGAAGCTTGGTACCGAGCTCGGATCCACTAGTCCAGTGTGGTGGAATTCTGCAGATATCAACAAGTTTGTACAAAAAAGCTGAACGAGAAACGTAAAATGATATAAATATCAATATATTAAATTAGATTTTGCATAAAAAACAGACTACATAATACTGTAAAACACAACATATCCAGTCACTATGGCGGCCGCATTAGGCACCCCAGGCTTTACACTTTATGCTTCCGGCTCGTATAATGTGTGGATTTTGAGTTAGGATCCGGCGAGATTTTCAGGAGCTAAGGAAGCTAAAATGGAGAAAAAAATCACTGGATATACCACCGTTGATATATCCCAATGGCATCGTAAAGAACATTTTGAGGCATTTCAGTCAGTTGCTCAATGTACCTATAACCAGACCGTTCAGCTGGATATTACGGCCTTTTTAAAGACCGTAAAGAAAAATAAGCACAAGTTTTATCCGGCCTTTATTCACATTCTTGCCCGCCTGATGAATGCTCATCCGGAATTCCGTATGGCAATGAAAGACGGTGAGCTGGTGATATGGGATAGTGTTCACCCTTGTTACACCGTTTTCCATGAGCAAACTGAAACGTTTTCATCGCTCTGGAGTGAATACCACGACGATTTCCGGCAGTTTCTACACATATATTCGCAAGATGTGGCGTGTTACGGTGAAAACCTGGCCTATTTCCCTAAAGGGTTTATTGAGAATATGTTTTTCGTCTCAGCCAATCCCTGGGTGAGTTTCACCAGTTTTGATTTAAACGTGGCCAATATGGACAACTTCTTCGCCCCCGTTTTCACCATGGGCAAATATTATACGCAAGGCGACAAGGTGCTGATGCCGCTGGCGATTCAGGTTCATCATGCCGTCTGTGATGGCTTCCATGTCGGCAGAATGCTTAATGAATTACAACAGTACTGCGATGAGTGGCAGGGCGGGGCGTAAAGATCTGGATCCGGCTTACTAAAAGCCAGATAACAGTATGCGTATTTGCGCGCTGATTTTTGCGGTATAAGAATATATACTGATATGTATACCCGAAGTATGTCAAAAAGAGGTGTGCTATGAAGCAGCGTATTACAGTGACAGTTGACAGCGACAGCTATCAGTTGCTCAAGGCATATATGATGTCAATATCTCCGGTCTGGTAAGCACAACCATGCAGAATGAAGCCCGTCGTCTGCGTGCCGAACGCTGGAAAGCGGAAAATCAGGAAGGGATGGCTGAGGTCGCCCGGTTTATTGAAATGAACGGCTCTTTTGCTGACGAGAACAGGGACTGGTGAAATGCAGTTTAAGGTTTACACCTATAAAAGAGAGAGCCGTTATCGTCTGTTTGTGGATGTACAGAGTGATATTATTGACACGCCCGGGCGACGGATGGTGATCCCCCTGGCCAGTGCACGTCTGCTGTCAGATAAAGTCTCCCGTGAACTTTACCCGGTGGTGCATATCGGGGATGAAAGCTGGCGCATGATGACCACCGATATGGCCAGTGTGCCGGTCTCCGTTATCGGGGAAGAAGTGGCTGATCTCAGCCACCGCGAAAATGACATCAAAAACGCCATTAACCTGATGTTCTGGGGAATATAAATGTCAGGCTCCGTTATACACAGCCAGTCTGCAGGTCGACCATAGTGACTGGATATGTTGTGTTTTACAGTATTATGTAGTCTGTTTTTTATGCAAAATCTAATTTAATATATTGATATTTATATCATTTTACGTTTCTCGTTCAGCTTTCTTGTACAAAGTGGTTGATATCCAGCACAGTGGCGGCCGCTCGAGTCTAGAGGGCCCGCGGTTCGAAGGTAAGCCTATCCCTAACCCTCTCCTCGGTCTCGATTCTACGCGTACCGGTTAGTAATGAGTTTGGAATTAATTCTGTGGAATGTGTGTCAGTTAGGGTGTGGAAAGTCCCCAGGCTCCCCAGGCAGGCAGAAGTATGCAAAGCATGCATCTCAATTAGTCAGCAACCAGGTGTGGAAAGTCCCCAGGCTCCCCAGCAGGCAGAAGTATGCAAAGCATGCATCTCAATTAGTCAGCAACCATAGTCCCGCCCCTAACTCCGCCCATCCCGCCCCTAACTCCGCCCAGTTCCGCCCATTCTCCGCCCCATGGCTGACTAATTTTTTTTATTTATGCAGAGGCCGAGGCCGCCTCTGCCTCTGAGCTATTCCAGAAGTAGTGAGGAGGCTTTTTTGGAGGCCTAGGCTTTTGCAAAAAGCTCCCGGGAGCTTGTATATCCATTTTCGGATCTGATCAGCACGTGTTGACAATTAATCATCGGCATAGTATATCGGCATAGTATAATACGACAAGGTGAGGAACTAAACCATGGCCAAGCCTTTGTCTCAAGAAGAATCCACCCTCATTGAAAGAGCAACGGCTACAATCAACAGCATCCCCATCTCTGAAGACTACAGCGTCGCCAGCGCAGCTCTCTCTAGCGACGGCCGCATCTTCACTGGTGTCAATGTATATCATTTTACTGGGGGACCTTGTGCAGAACTCGTGGTGCTGGGCACTGCTGCTGCTGCGGCAGCTGGCAACCTGACTTGTATCGTCGCGATCGGAAATGAGAACAGGGGCATCTTGAGCCCCTGCGGACGGTGCCGACAGGTGCTTCTCGATCTGCATCCTGGGATCAAAGCCATAGTGAAGGACAGTGATGGACAGCCGACGGCAGTTGGGATTCGTGAATTGCTGCCCTCTGGTTATGTGTGGGAGGGCTAAGCACAATTCGAGCTCGGTACCTTTAAGACCAATGACTTACAAGGCAGCTGTAGATCTTAGCCACTTTTTAAAAGAAAAGGGGGGACTGGAAGGGCTAATTCACTCCCAACGAAGACAAGATCTGCTTTTTGCTTGTACTGGGTCTCTCTGGTTAGACCAGATCTGAGCCTGGGAGCTCTCTGGCTAACTAGGGAACCCACTGCTTAAGCCTCAATAAAGCTTGCCTTGAGTGCTTCAAGTAGTGTGTGCCCGTCTGTTGTGTGACTCTGGTAACTAGAGATCCCTCAGACCCTTTTAGTCAGTGTGGAAAATCTCTAGCAGTAGTAGTTCATGTCATCTTATTATTCAGTATTTATAACTTGCAAAGAAATGAATATCAGAGAGTGAGAGGAACTTGTTTATTGCAGCTTATAATGGTTACAAATAAAGCAATAGCATCACAAATTTCACAAATAPAGCATTTTTTTCACTGCATTCTAGTTGTGGTTTGTCCAAACTCATCAATGTATCTTATCATGTCTGGCTCTAGCTATCCCGCCCCTAACTCCGCCCATCCCGCCCCTAACTCCGCCCAGTTCCGCCCATTCTCCGCCCCATGGCTGACTAATTTTTTTTATTTATGCAGAGGCCGAGGCCGCCTCGGCCTCTGAGCTATTCCAGAAGTAGTGAGGAGGCTTTTTTGGAGGCCTAGGGACGTACCCAATTCGCCCTATAGTGAGTCGTATTACGCGCGCTCACTGGCCGTCGTTTTACAACGTCGTGACTGGGAAAACCCTGGCGTTACCCAACTTAATCGCCTTGCAGCACATCCCCCTTTCGCCAGCTGGCGTAATAGCGAAGAGGCCCGCACCGATCGCCCTTCCCAACAGTTGCGCAGCCTGAATGGCGAATGGGACGCGCCCTGTAGCGGCGCATTAAGCGCGGCGGGTGTGGTGGTTACGCGCAGCGTGACCGCTACACTTGCCAGCGCCCTAGCGCCCGCTCCTTTCGCTTTCTTCCCTTCCTTTCTCGCCACGTTCGCCGGCTTTCCCCGTCAAGCTCTAAATCGGGGGCTCCCTTTAGGGTTCCGATTTAGTGCTTTACGGCACCTCGACCCCAAAAAACTTGATTAGGGTGATGGTTCACGTAGTGGGCCATCGCCCTGATAGACGGTTTTTCGCCCTTTGACGTTGGAGTCCACGTTCTTTAATAGTGGACTCTTGTTCCAAACTGGAACAACACTCAACCCTATCTCGGTCTATTCTTTTGATTTATAAGGGATTTTGCCGATTTCGGCCTATTGGTTAAAAAATGAGCTGATTTAACAAAAATTTAACGCGAATTTTAACAAAATATTAACGCTTACAATTTAGGTGGCACTTTTCGGGGAAATGTGCGCGGAACCCCTATTTGTTTATTTTTCTAAATACATTCAAATATGTATCCGCTCATGAGACAATAACCCTGATAAATGCTTCAATAATATTGAAAAAGGAAGAGTATGAGTATTCAACATTTCCGTGTCGCCCTTATTCCCTTTTTTGCGGCATTTTGCCTTCCTGTTTTTGCTCACCCAGAAACGCTGGTGAAAGTAAAAGATGCTGAAGATCAGTTGGGTGCACGAGTGGGTTACATCGAACTGGATCTCAACAGCGGTAAGATCCTTGAGAGTTTTCGCCCCGAAGAACGTTTTCCAATGATGAGCACTTTTAAAGTTCTGCTATGTGGCGCGGTATTATCCCGTATTGACGCCGGGCAAGAGCAACTCGGTCGCCGCATACACTATTCTCAGAATGACTTGGTTGAGTACTCACCAGTCACAGAAAAGCATCTTACGGATGGCATGACAGTAAGAGAATTATGCAGTGCTGCCATAACCATGAGTGATAACACTGCGGCCAACTTACTTCTGACAACGATCGGAGGACCGAAGGAGCTAACCGCTTTTTTGCACAACATGGGGGATCATGTAACTCGCCTTGATCGTTGGGAACCGGAGCTGAATGAAGCCATACCAAACGACGAGCGTGACACCACGATGCCTGTAGCAATGGCAACAACGTTGCGCAAACTATTAACTGGCGAACTACTTACTCTAGCTTCCCGGCAACAATTAATAGACTGGATGGAGGCGGATAAAGTTGCAGGACCACTTCTGCGCTCGGCCCTTCCGGCTGGCTGGTTTATTGCTGATAAATCTGGAGCCGGTGAGCGTGGGTCTCGCGGTATCATTGCAGCACTGGGGCCAGATGGTAAGCCCTCCCGTATCGTAGTTATCTACACGACGGGGAGTCAGGCAACTATGGATGAACGAAATAGACAGATCGCTGAGATAGGTGCCTCACTGATTAAGCATTGGTAACTGTCAGACCAAGTTTACTCATATATACTTTAGATTGATTTAAAACTTCATTTTTAATTTAAAAGGATCTAGGTGAAGATCCTTTTTGATAATCTCATGACCAAAATCCCTTAACGTGAGTTTTCGTTCCACTGAGCGTCAGACCCCGTAGAAAAGATCAAAGGATCTTCTTGAGATCCTTTTTTTCTGCGCGTAATCTGCTGCTTGCAAACAAAAAAACCACCGCTACCAGCGGTGGTTTGTTTGCCGGATCAAGAGCTACCAACTCTTTTTCCGAAGGTAACTGGCTTCAGCAGAGCGCAGATACCAAATACTGTTCTTCTAGTGTAGCCGTAGTTAGGCCACCACTTCAAGAACTCTGTAGCACCGCCTACATACCTCGCTCTGCTAATCCTGTTACCAGTGGCTGCTGCCAGTGGCGATAAGTCGTGTCTTACCGGGTTGGACTCAAGACGATAGTTACCGGATAAGGCGCAGCGGTCGGGCTGAACGGGGGGTTCGTGCACACAGCCCAGCTTGGAGCGAACGACCTACACCGAACTGAGATACCTACAGCGTGAGCTATGAGAAAGCGCCACGCTTCCCGAAGGGAGAAAGGCGGACAGGTATCCGGTAAGCGGCAGGGTCGGAACAGGAGAGCGCACGAGGGAGCTTCCAGGGGGAAACGCCTGGTATCTTTATAGTCCTGTCGGGTTTCGCCACCTCTGACTTGAGCGTCGATTTTTGTGATGCTCGTCAGGGGGGCGGAGCCTATGGAAAAACGCCAGCAACGCGGCCTTTTTACGGTTCCTGGCCTTTTGCTGGCCTTTTGCTCACATGTTCTTTCCTGCGTTATCCCCTGATTCTGTGGATAACCGTATTACCGCCTTTGAGTGAGCTGATACCGCTCGCCGCAGCCGAACGACCGAGCGCAGCGAGTCAGTGAGCGAGGAAGCGGAAGAGCGCCCAATACGCAAACCGCCTCTCCCCGCGCGTTGGCCGATTCATTAATGCAGCTGGCACGACAGGTTTCCCGACTGGAAAGCGGGCAGTGAGCGCAACGCAATTAATGTGAGTTAGCTCACTCATTAGGCACCCCAGGCTTTACACTTTATGCTTCCGGCTCGTATGTTGTGTGGAATTGTGAGCGGATAACAATTTCACACAGGAAACAGCTATGACCATGATTACGCCAAGCGCGCAATTAACCCTCACTAAAGGGAACAAAAGCTGGAGCTGCAAGCTT

[1253] TABLE 21 Nucleotide sequence of plasmid pLP1.TTGGCCCATTGCATACGTTGTATCCATATCATAATATGTACATTTATATTGGCTCATGTCCAACATTACCGCCATGTTGACATTGATTATTGACTAGTTATTAATAGTAATCAATTACGGGGTCATTAGTTCATAGCCCATATATGGAGTTCCGCGTTACATAACTTACGGTAAATGGCCCGCCTGGCTGACCGCCCAACGACCCCCGCCCATTGACGTCAATAATGACGTATGTTCCCATAGTAACGCCAATAGGGACTTTCCATTGACGTCAATGGGTGGAGTATTTACGGTAAACTGCCCACTTGGCAGTACATCAAGTGTATCATATGCCAAGTACGCCCCCTATTGACGTCAATGACGGTAAATGGCCCGCCTGGCATTATGCCCAGTACATGACCTTATGGGACTTTCCTACTTGGCAGTACATCTACGTATTAGTCATCGCTATTACCATGGTGATGCGGTTTTGGCAGTACATCAATGGGCGTGGATAGCGGTTTGACTCACGGGGATTTCCAAGTCTCCACCCCATTGACGTCAATGGGAGTTTGTTTTGGCACCAAAATCAACGGCACTTTCCAAAATGTCGTAACAACTCCGCCCCATTGACGCAAATGGGCGGTAGGCGTGTACGGTGGGAGGTCTATATAAGCAGAGCTCGTTTAGTGAACCGTCAGATCGCCTGGAGACGCCATCCACGCTGTTTTGACCTCCATAGAAGACACCGGGACCGATCCAGCCTCCCCTCGAAGCTTACATGTGGTACCGAGCTCGGATCCTGAGAACTTCAGGGTGAGTCTATGGGACCCTTGATGTTTTCTTTCCCCTTCTTTTCTATGGTTAAGTTCATGTCATAGGAAGGGGAGAAGTAACAGGGTACACATATTGACCAAATCAGGGTAATTTTGCATTTGTAATTTTAAAAAATGCTTTCTTCTTTTAATATACTTTTTTGTTTATCTTATTTCTAATACTTTCCCTAATCTCTTTCTTTCAGGGCAATAATGATACAATGTATCATGCCTCTTTGCACCATTCTAAAGAATAACAGTGATAATTTCTGGGTTAAGGCAATAGCAATATTTCTGCATATAAATATTTCTGCATATAAATTGTAACTGATGTAAGAGGTTTCATATTGCTAATAGCAGCTACAATCCAGCTACCATTCTGCTTTTATTTTATGGTTGGGATAAGGCTGGATTATTCTGAGTCCAAGCTAGGCCCTTTTGCTAATCATGTTCATACCTCTTATCTTCCTCCCACAGCTCCTGGGCAACGTGCTGGTCTGTGTGCTGGCCCATCACTTTGGCAAAGCACGTGAGATCTGAATTCGAGATCTGCCGCCGCCATGGGTGCGAGAGCGTCAGTATTAAGCGGGGGAGAATTAGATCGATGGGAAAAAATTCGGTTAAGGCCAGGGGGAAAGAAAAAATATAAATTAAAACATATAGTATGGGCAAGCAGGGAGCTAGAACGATTCGCAGTTAATCCTGGCCTGTTAGAAACATCAGAAGGCTGTAGACAAATACTGGGACAGCTACAACCATCCCTTCAGACAGGATCAGAAGAACTTAGATCATTATATAATACAGTAGCAACCCTCTATTGTGTGCATCAAAGGATAGAGATAAAAGACACCAAGGAAGCTTTAGACAAGATAGAGGAAGAGCAAAACAAAAGTAAGAAAAAAGCACAGCAAGCAGCAGCTGACACAGGACACAGCAATCAGGTCAGCCAAAATTACCCTATAGTGCAGAACATCCAGGGGCAAATGGTACATCAGGCCATATCACCTAGAACTTTAAATGCATGGGTAAAAGTAGTAGAAGAGAAGGCTTTCAGCCCAGAAGTGATACCCATGTTTTCAGCATTATCAGAAGGAGCCACCCCACAAGATTTAAACACCATGCTAAACACAGTGGGGGGACATCAAGCAGCCATGCAAATGTTAAAAGAGACCATCAATGAGGAAGCTGCAGAATGGGATAGAGTGCATCCAGTGCATGCAGGGCCTATTGCACCAGGCCAGATGAGAGAACCAAGGGGAAGTGACATAGCAGGAACTACTAGTACCCTTCAGGAACAAATAGGATGGATGACACATAATCCACCTATCCCAGTAGGAGAAATCTATAAAAGATGGATAATCCTGGGATTAAATAAAATAGTAAGAATGTATAGCCCTACCAGCATTCTGGACATAAGACAAGGACCAAAGGAACCCTTTAGAGACTATGTAGACCGATTCTATAAAACTCTAAGAGCCGAGCAAGCTTCACAAGAGGTAAAAAATTGGATGACAGAAACCTTGTTGGTCCAAAATGCGAACCCAGATTGTAAGACTATTTTAAAAGCATTGGGACCAGGAGCGACACTAGAAGAAATGATGACAGCATGTCAGGGAGTGGGGGGACCCGGCCATAAAGCAAGAGTTTTGGCTGAAGCAATGAGCCAAGTAACAAATCCAGCTACCATAATGATACAGAAAGGCAATTTTAGGAACCAAAGAAAGACTGTTAAGTGTTTCAATTGTGGCAAAGAAGGGCACATAGCCAAAAATTGCAGGGCCCCTAGGAAAAAGGGCTGTTGGAAATGTGGAAAGGAAGGACACCAAATGAAAGATTGTACTGAGAGACAGGCTAATTTTTTAGGGAAGATCTGGCCTTCCCACAAGGGAAGGCCAGGGAATTTTCTTCAGAGCAGACCAGAGCCAACAGCCCCACCAGAAGAGAGCTTCAGGTTTGGGGAAGAGACAACAACTCCCTCTCAGAAGCAGGAGCCGATAGACAAGGAACTGTATCCTTTAGCTTCCCTCAGATCACTCTTTGGCAGCGACCCCTCGTCACAATAAAGATAGGGGGGCAATTAAAGGAAGCTCTATTAGATACAGGAGCAGATGATACAGTATTAGAAGAAATGAATTTGCCAGGAAGATGGAAACCAAAAATGATAGGGGGAATTGGAGGTTTTATCAAAGTAAGACAGTATGATCAGATACTCATAGAAATCTGCGGACATAAAGCTATAGGTACAGTATTAGTAGGACCTACACCTGTCAACATAATTGGAAGAAATCTGTTGACTCAGATTGGCTGCACTTTAAATTTTCCCATTAGTCCTATTGAGACTGTACCAGTAAAATTAAAGCCAGGAATGGATGGCCCAAAAGTTAAACAATGGCCATTGACAGAAGAAAAAATAAAAGCATTAGTAGAAATTTGTACAGAAATGGAAAAGGAAGGAAAAATTTCAAAAATTGGGCCTGAAAATCCATACAATACTCCAGTATTTGCCATAAAGAAAAAAGACAGTACTAAATGGAGAAAATTAGTAGATTTCAGAGAACTTAATAAGAGAACTCAAGATTTCTGGGAAGTTCAATTAGGAATACCACATCCTGCAGGGTTAAAACAGAAAAAATCAGTAACAGTACTGGATGTGGGCGATGCATATTTTTCAGTTCCCTTAGATAAAGACTTCAGGAAGTATACTGCATTTACCATACCTAGTATAAACAATGAGACACCAGGGATTAGATATCAGTACAATGTGCTTCCACAGGGATGGAAAGGATCACCAGCAATATTCCAGTGTAGCATGACAAAAATCTTAGAGCCTTTTAGAAAACAAAATCCAGACATAGTCATCTATCAATACATGGATGATTTGTATGTAGGATCTGACTTAGAAATAGGGCAGCATAGAACAAAAATAGAGGAACTGAGACAACATCTGTTGAGGTGGGGATTTACCACACCAGACAAAAAACATCAGAAAGAACCTCCATTCCTTTGGATGGGTTATGAACTCCATCCTGATAAATGGACAGTACAGCCTATAGTGCTGCCAGAAAAGGACAGCTGGACTGTCAATGACATACAGAAATTAGTGGGAAAATTGAATTGGGCAAGTCAGATTTATGCAGGGATTAAAGTAAGGCAATTATGTAAACTTCTTAGGGGAACCAAAGCACTAACAGAAGTAGTACCACTAACAGAAGAAGCAGAGCTAGAACTGGCAGAAAACAGGGAGATTCTAAAAGAACCGGTACATGGAGTGTATTATGACCCATCAAAAGACTTAATAGCAGAAATACAGAAGCAGGGGCAAGGCCAATGGACATATCAAATTTATCAAGAGCCATTTAAAAATCTGAAAACAGGAAAGTATGCAAGAATGAAGGGTGCCCACACTAATGATGTGAAACAATTAACAGAGGCAGTACAAAAAATAGCCACAGAAAGCATAGTAATATGGGGAAAGACTCCTAAATTTAAATTACCCATACAAAAGGAAACATGGGAAGCATGGTGGACAGAGTATTGGCAAGCCACCTGGATTCCTGAGTGGGAGTTTGTCAATACCCCTCCCTTAGTGAAGTTATGGTACCAGTTAGAGAAAGAACCCATAATAGGAGCAGAAACTTTCTATGTAGATGGGGCAGCCAATAGGGAAACTAAATTAGGAAAAGCAGGATATGTAACTGACAGAGGAAGACAAAAAGTTGTCCCCCTAACGGACACAACAAATCAGAAGACTGAGTTACAAGCAATTCATCTAGCTTTGCAGGATTCGGGATTAGAAGTAAACATAGTGACAGACTCACAATATGCATTGGGAATCATTCAAGCACAACCAGATAAGAGTGAATCAGAGTTAGTCAGTCAAATAATAGAGCAGTTAATAAAAAAGGAAAAAGTCTACCTGGCATGGGTACCAGCACACAAAGGAATTGGAGGAAATGAACAAGTAGATAAATTGGTCAGTGCTGGAATCAGGAAAGTACTATTTTTAGATGGAATAGATAAGGCCCAAGAAGAACATGAGAAATATCACAGTAATTGGAGAGCPATGGCTAGTGATTTTAACCTACCACCTGTAGTAGCAAAAGAAATAGTAGCCAGCTGTGATAAATGTCAGCTAAAAGGGGAAGCCATGCATGGACAAGTAGACTGTAGCCCAGGAATATGGCAGCTAGATTGTACACATTTAGAAGGAAAAGTTATCTTGGTAGCAGTTCATGTAGCCAGTGGATATATAGAAGCAGAAGTAATTCCAGCAGAGACAGGGCAAGAAACAGCATACTTCCTCTTAAAATTAGCAGGAAGATGGCCAGTAAAAACAGTACATACAGACAATGGCAGCAATTTCACCAGTACTACAGTTAAGGCCGCCTGTTGGTGGGCGGGGATCAAGCAGGAATTTGGCATTCCCTACAATCCCCAAAGTCAAGGAGTAATAGAATCTATGAATAAAGAATTAAAGAAAATTATAGGACAGGTAAGAGATCAGGCTGAACATCTTAAGACAGCAGTACAAATGGCAGTATTCATCCACAATTTTAAAAGAAAAGGGGGGATTGGGGGGTACAGTGCAGGGGAAAGAATAGTAGACATAATAGCAACAGACATACAAACTAAAGAATTACAAAAACAAATTACAAAAATTCAAAATTTTCGGGTTTATTACAGGGACAGCAGAGATCCAGTTTGGAAAGGACCAGCAAAGCTCCTCTGGAAAGGTGAAGGGGCAGTAGTAATACAAGATAATAGTGACATAAAAGTAGTGCCAAGAAGAAAAGCAAAGATCATCAGGGATTATGGAAAACAGATGGCAGGTGATGATTGTGTGGCAAGTAGACAGGATGAGGATTAACACATGGAATTCCGGAGCGGCCGCAGGAGCTTTGTTCCTTGGGTTCTTGGGAGCAGCAGGAAGCACTATGGGCGCAGCGTCAATGACGCTGACGGTACAGGCCAGACAATTATTGTCTGGTATAGTGCAGCAGCAGAACAATTTGCTGAGGGCTATTGAGGCGCAACAGCATCTGTTGCAACTCACAGTCTGGGGCATCAAGCAGCTCCAGGCAAGAATCCTGGCTGTGGAAAGATACCTAAAGGATCAACAGCTCCTGGGGATTTGGGGTTGCTCTGGAAAACTCATTTGCACCACTGCTGTGCCTTGGAATGCTAGTTGGAGTAATAAATCTCTGGAACAGATTTGGAATCACACGACCTGGATGGAGTGGGACAGAGAAATTAACAATTACACAAGCTTCCGCGGAATTCACCCCACCAGTGCAGGCTGCCTATCAGAAAGTGGTGGCTGGTGTGGCTAATGCCCTGGCCCACAAGTATCACTAAGCTCGCTTTCTTGCTGTCCAATTTCTATTAAAGGTTCCTTTGTTCCCTAAGTCCAACTACTAAACTGGGGGATATTATGAAGGGCCTTGAGCATCTGGATTCTGCCTAATAAAAAACATTTATTTTCATTGCAATGATGTATTTAAATTATTTCTGAATATTTTACTAAAAAGGGAATGTGGGAGGTCAGTGCATTTAAAACATAAAGAAATGAAGAGCTAGTTCAAACCTTGGGAAAATACACTATATCTTAAACTCCATGAAAGAAGGTGAGGCTGCAAACAGCTAATGcACATTGGCAACAGCCCCTGATGCCTATGCCTTATTCATCCCTCAGAAAAGGATTCAAGTAGAGGCTTGATTTGGAGGTTAAAGTTTTGCTATGCTGTATTTTACATTACTTATTGTTTTAGCTGTCCTCATGAATGTCTTTTCACTACCCATTTGCTTATCCTGCATCTCTCAGCCTTGACTCCACTCAGTTCTCTTGCTTAGAGATACCACCTTTCCCCTGAAGTGTTCCTTCCATGTTTTACGGCGAGATGGTTTCTCCTCGCCTGGCCACTCAGCCTTAGTTGTCTCTGTTGTCTTATAGAGGTCTACTTGAAGAAGGAAAAACAGGGGGCATGGTTTGACTGTCCTGTGAGCCCTTCTTCCCTGCCTCCCCCACTCACAGTGACCCGGAATCCCTCGACATGGCAGTCTAGCACTAGTGCGGCCGCAGATCTGCTTCCTCGCTCACTGACTCGCTGCGCTCGGTCGTTCGGCTGCGGCGAGCGGTATCAGCTCACTCAAAGGCGGTAATACGGTTATCCACAGAATCAGGGGATAACGCAGGAAAGAACATGTGAGCAAAAGGCCAGCAAAAGGCCAGGAACCGTAAAAAGGCCGCGTTGCTGGCGTTTTTCCATAGGCTCCGCCCCCCTGACGAGCATCACAAAAATCGACGCTCAAGTCAGAGGTGGCGAAACCCGACAGGACTATAAAGATACCAGGCGTTTCCCCCTGGAAGCTCCCTCGTGCGCTCTCCTGTTCCGACCCTGCCGCTTACCGGATACCTGTCCGCCTTTCTCCCTTCGGGAAGCGTGGCGCTTTCTCATAGCTCACGCTGTAGGTATCTCAGTTCGGTGTAGGTCGTTCGCTCCAAGCTGGGCTGTGTGCACGAACCCCCCGTTCAGCCCGACCGCTGCGCCTTATCCGGTAACTATCGTCTTGAGTCCAACCCGGTAAGACACGACTTATCGCCACTGGCAGCAGCCACTGGTAACAGGATTAGCAGAGCGAGGTATGTAGGCGGTGCTACAGAGTTCTTGAAGTGGTGGCCTAACTACGGCTACACTAGAAGAACAGTATTTGGTATCTGCGCTCTGCTGAAGCCAGTTACCTTCGGAAAAAGAGTTGGTAGCTCTTGATCCGGCAAACAAACCACCGCTGGTAGCGGTGGTTTTTTTGTTTGCAAGCAGCAGATTACGCGCAGAAAAAAAGGATCTCAAGAAGATCCTTTGATCTTTTCTACGGGGTCTGACGCTCAGTGGAACGAAAACTCACGTTAAGGGATTTTGGTCATGAGATTATCAAAAAGGATCTTCACCTAGATCCTTTTAAATTAAAAATGAAGTTTTAAATCAATCTAAAGTATATATGAGTAAACTTGGTCTGACAGTTACCAATGCTTAATCAGTGAGGCACCTATCTCAGCGATCTGTCTATTTCGTTCATCCATAGTTGCCTGACTCCCCGTCGTGTAGATAACTACGATACGGGAGGGCTTACCATCTGGCCCCAGTGCTGCAATGATACCGCGAGACCCACGCTCACCGGCTCCAGATTTATCAGCAATAAACCAGCCAGCCGGAAGGGCCGAGCGCAGAAGTGGTCCTGCAACTTTATCCGCCTCCATCCAGTCTATTAATTGTTGCCGGGAAGCTAGAGTAAGTAGTTCGCCAGTTAATAGTTTGCGCAACGTTGTTGCCATTGCTACAGGCATCGTGGTGTCACGCTCGTCGTTTGGTATGGCTTCATTCAGCTCCGGTTCCCAACGATCAAGGCGAGTTACATGATCCCCCATGTTGTGCAAAAAAGCGGTTAGCTCCTTCGGTCCTCCGATCGTTGTCAGAAGTAAGTTGGCCGCAGTGTTATCACTCATGGTTATGGCAGCACTGCATAATTCTCTTACTGTCATGCCATCCGTAAGATGCTTTTCTGTGACTGGTGAGTACTCAACCAAGTCATTCTGAGAATAGTGTATGCGGCGACCGAGTTGCTCTTGCCCGGCGTCAATACGGGATAATACCGCGCCACATAGCAGAACTTTAAAAGTGCTCATCATTGGAAAACGTTCTTCGGGGCGAAAACTCTCAAGGATCTTACCGCTGTTGAGATCCAGTTCGATGTAACCCACTCGTGCACCCAACTGATCTTCAGCATCTTTTACTTTCACCAGCGTTTCTGGGTGAGCAAAAACAGGAAGGCAAAATGCCGCAAAAAAGGGAATAAGGGCGACACGGAAATGTTGAATACTCATACTCTTCCTTTTTCAATATTATTGAAGCATTTATCAGGGTTATTGTCTCATGAGCGGATACATATTTGAATGTATTTAGAAAAATAAACAAATAGGGGTTCCGCGCACATTTCCCCGAAAAGTGCCACCTGACGGGATCCCCTGAGGGGGCCCCCATGGGCTAGAGGATCCGGCCTCGGCCTCTGCATAAATAAAAAAAATTAGTCAGCCATGAGC

[1254] TABLE 22 Nucleotide sequence of plasmid pLP2.AATGTAGTCTTATGCAATACTCTTGTAGTCTTGCAACATGGTAACGATGAGTTAGCAACATGCCTTACAAGGAGAGAAAAAGCACCGTGCATGCCGATTGGTGGAAGTAAGGTGGTACGATCGTGCCTTATTAGGAAGGCAACAGACGGGTCTGACATGGATTGGACGAACCACTGAATTCCGCATTGCAGAGATATTGTATTTAAGTGCCTAGCTCGATACAATAAACGCCATTTGACCATTCACCACATTGGTGTGCACCTCCAAGCTCGAGCTCGTTTAGTGAACCGTCAGATCGCCTGGAGACGCCATCCACGCTGTTTTGACCTCCATAGAAGACACCGGGACCGATCCAGCCTCCCCTCGAAGCTAGTCGATTAGGCATCTCCTATGGCAGGAAGAAGCGGAGACAGCGACGAAGACCTCCTCAAGGCAGTCAGACTCATCAAGTTTCTCTATCAAAGCAACCCACCTCCCAATCCCGAGGGGACCCGACAGGCCCGAAGGAATAGAAGAAGAAGGTGGAGAGAGAGACAGAGACAGATCCATTCGATTAGTGAACGGATCCTTAGCACTTATCTGGGACGATCTGCGGAGCCTGTGCCTCTTCAGCTACCACCGCTTGAGAGACTTACTCTTGATTGTAACGAGGATTGTGGAACTTCTGGGACGCAGGGGGTGGGAAGCCCTCAAATATTGGTGGAATCTCCTACAATATTGGAGTCAGGAGCTAAAGAATAGTGCTGTTAGCTTGCTCAATGCCACAGCTATAGCAGTAGCTGAGGGGACAGATAGGGTTATAGAAGTAGTACAAGAAGCTTGGCACTGGCCGTCGTTTTACAACGTCGTGATCTGAGCCTGGGAGATCTCTGGCTAACTAGGGAACCCACTGCTTAAGCCTCAATAAAGCTTGCCTTGAGTGCTTCAAGTAGTGTGTGCCCGTCTGTTGTGTGACTCTGGTAACTAGAGATCAGGAAAACCCTGGCGTTACCCAACTTAATCGCCTTGCAGCACATCCCCCTTTCGCCAGCTGGCGTAATAGCGAAGAGGCCCGCACCGATCGCCCTTCCCAACAGTTGCGCAGCCTGAATGGCGAATGGCGCCTGATGCGGTATTTTCTCCTTACGCATCTGTGCGGTATTTCACACCGCATACGTCAAAGCAACCATAGTACGCGCCCTGTAGCGGCGCATTAAGCGCGGCGGGTGTGGTGGTTACGCGCAGCGTGACCGCTACACTTGCCAGCGCCCTAGCGCCCGCTCCTTTCGCTTTCTTCCCTTCCTTTCTCGCCACGTTCGCCGGCTTTCCCCGTCAAGCTCTAAATCGGGGGCTCCCTTTAGGGTTCCGATTTAGTGCTTTACGGCACCTCGACCCCAAAAAACTTGATTTGGGTGATGGTTCACGTAGTGGGCCATCGCCCTGATAGACGGTTTTTCGCCCTTTGACGTTGGAGTCCACGTTCTTTAATAGTGGACTCTTGTTCCAAACTGGAACAACACTCAACCCTATCTCGGGCTATTCTTTTGATTTATAAGGGATTTTGCCGATTTCGGCCTATTGGTTAAAAAATGAGCTGATTTAACAAAAATTTAACGCGAATTTTAACAAAATATTAACGTTTACAATTTTATGGTGCACTCTCAGTACAATCTGCTCTGATGCCGCATAGTTAAGCCAGCCCCGACACCCGCCAACACCCGCTGACGCGCCCTGACGGGCTTGTCTGCTCCCGGCATCCGCTTACAGACAAGCTGTGACCGTCTCCGGGAGCTGCATGTGTCAGAGGTTTTCACCGTCATCACCGAAACGCGCGAGACGAAAGGGCCTCGTGATACGCCTATTTTTATAGGTTAATGTCATGATAATAATGGTTTCTTAGACGTCAGGTGGCACTTTTCGGGGAAATGTGCGCGGAACCCCTATTTGTTTATTTTTCTAAATACATTCAAATATGTATCCGCTCATGAGACAATAACCCTGATAAATGCTTCAATAATATTGAAAAAGGAAGAGTATGAGTATTCAACATTTCCGTGTCGCCCTTATTCCCTTTTTTGCGGCATTTTGCCTTCCTGTTTTTGCTCACCCAGAAACGCTGGTGAAAGTAAAAGATGCTGAAGATCAGTTGGGTGCACGAGTGGGTTACATCGAACTGGATCTCAACAGCGGTAAGATCCTTGAGAGTTTTCGCCCCGAAGAACGTTTTCCAATGATGAGCACTTTTAAAGTTCTGCTATGTGGCGCGGTATTATCCCGTATTGACGCCGGGCAAGAGCAACTCGGTCGCCGCATACACTATTCTCAGAATGACTTGGTTGAGTACTCACCAGTCACAGAAAAGCATCTTACGGATGGCATGACAGTAAGAGAATTATGCAGTGCTGCCATAACCATGAGTGATAACACTGCGGCCAACTTACTTCTGACAACGATCGGAGGACCGAAGGAGCTAACCGCTTTTTTGCACAACATGGGGGATCATGTAACTCGCCTTGATCGTTGGGAACCGGAGCTGAATGAAGCCATACCAAACGACGAGCGTGACACCACGATGCCTGTAGCAATGGCAACAACGTTGCGCAAACTATTAACTGGCGAACTACTTACTCTAGCTTCCCGGCAACAATTAATAGACTGGATGGAGGCGGATAAAGTTGCAGGACCACTTCTGCGCTCGGCCCTTCCGGCTGGCTGGTTTATTGCTGATAAATCTGGAGCCGGTGAGCGTGGGTCTCGCGGTATCATTGCAGCACTGGGGCCAGATGGTAAGCCCTCCCGTATCGTAGTTATCTACACGACGGGGAGTCAGGCAACTATGGATGAACGAAATAGACAGATCGCTGAGATAGGTGCCTCACTGATTAAGCATTGGTAACTGTCAGACCAAGTTTACTCATATATACTTTAGATTGATTTAAAACTTCATTTTTAATTTAAAAGGATCTAGGTGAAGATCCTTTTTGATAATCTCATGACCAAAATCCCTTAACGTGAGTTTTCGTTCCACTGAGCGTCAGACCCCGTAGAAAAGATCAAAGGATCTTCTTGAGATCCTTTTTTTCTGCGCGTAATCTGCTGCTTGCAAACAAAAAAACCACCGCTACCAGCGGTGGTTTGTTTGCCGGATCAAGAGCTACCAACTCTTTTTCCGAAGGTAACTGGCTTCAGCAGAGCGCAGATACCAAATACTGTTCTTCTAGTGTAGCCGTAGTTAGGCCACCACTTCAAGAACTCTGTAGCACCGCCTACATACCTCGCTCTGCTAATCCTGTTACCAGTGGCTGCTGCCAGTGGCGATAAGTCGTGTCTTACCGGGTTGGACTCAAGACGATAGTTACCGGATAAGGCGCAGCGGTCGGGCTGAACGGGGGGTTCGTGCACACAGCCCAGCTTGGAGCGAACGACCTACACCGAACTGAGATACCTACAGCGTGAGCTATGAGAAAGCGCCACGCTTCCCGAAGGGAGAAAGGCGGACAGGTATCCGGTAAGCGGCAGGGTCGGAACAGGAGAGCGCACGAGGGAGCTTCCAGGGGGAAACGCCTGGTATCTTTATAGTCCTGTCGGGTTTCGCCACCTCTGACTTGAGCGTCGATTTTTGTGATGCTCGTCAGGGGGGCGGAGCCTATGGAAAAACGCCAGCAACGCGGCCTTTTTACGGTTCCTGGCCTTTTGCTGGCCTTTTGCTCACATGTTCTTTCCTGCGTTATCCCCTGATTCTGTGGATAACCGTATTACCGCCTTTGAGTGAGCTGATACCGCTCGCCGCAGCCGAACGACCGAGCGCAGCGAGTCAGTGAGCGAGGAAGCGGAAGAGCGCCCAATACGCAAACCGCCTCTCCCCGCGCGTTGGCCGATTCATTAATGCAGCTGGCACGACAGGTTTCCCGACTGGAAAGCGGGCAGTGAGCGCAACGCAATTAATGTGAGTTAGCTCACTCATTAGGCACCCCAGGCTTTACACTTTATGCTTCCGGCTCGTATGTTGTGTGGAATTGTGAGCGGATAACAATTTCACACAGGAAACAGCTATGACATGATTACGAATTCGATGTACGGGCCAGATATACGCGTATCTGAGGGGACTAGGGTGTGTTTAGGCGAAAAGCGGGGCTTCGGTTGTACGCGGTTAGGAGTCCCCTCAGGATATAGTAGTTTCGCTTTTGCATAGGGAGGGGGA

[1255] TABLE 23 Nucleotide sequence of plasmid pLP/VSVG.TTGGCCCATTGCATACGTTGTATCCATATCATAATATGTACATTTATATTGGCTCATGTCCAACATTACCGCCATGTTGACATTGATTATTGACTAGTTATTAATAGTAATCAATTACGGGGTCATTAGTTCATAGCCCATATATGGAGTTCCGCGTTACATAACTTACGGTAAATGGCCCGCCTGGCTGACCGCCCAACGACCCCCGCCCATTGACGTCAATAATGACGTATGTTCCCATAGTAACGCCAATAGGGACTTTCCATTGACGTCAATGGGTGGAGTATTTACGGTAAACTGCCCACTTGGCAGTACATCAAGTGTATCATATGCCAAGTACGCCCCCTATTGACGTCAATGACGGTAAATGGCCCGCCTGGCATTATGCCCAGTACATGACCTTATGGGACTTTCCTACTTGGCAGTACATCTACGTATTAGTCATCGCTATTACCATGGTGATGCGGTTTTGGCAGTACATCAATGGGCGTGGATAGCGGTTTGACTCACGGGGATTTCCAAGTCTCCACCCCATTGACGTCAATGGGAGTTTGTTTTGGCACCAAAATCAACGGGACTTTCCAAAATGTCGTAACAACTCCGCCCCATTGACGCAAATGGGCGGTAGGCGTGTACGGTGGGAGGTCTATATAAGCAGAGCTCGTTTAGTGAACCGTCAGATCGCCTGGAGACGCCATCCACGCTGTTTTGACCTCCATAGAAGACACCGGGACCGATCCAGCCTCCCCTCGAAGCTTACATGTGGTACCGAGCTCGGATCCTGAGAACTTCAGGGTGAGTCTATGGGACCCTTGATGTTTTCTTTCCCCTTCTTTTCTATGGTTAAGTTCATGTCATAGGAAGGGGAGAAGTAACAGGGTACACATATTGACCAAATCAGGGTAATTTTGCATTTGTAATTTTAAAAAATGCTTTCTTCTTTTAATATACTTTTTTGTTTATCTTATTTCTAATACTTTCCCTAATCTCTTTCTTTCAGGGCAATAATGATACAATGTATCATGCCTCTTTGCACCATTCTAAAGAATAACAGTGATAATTTCTGGGTTAAGGCAATAGCAATATTTCTGCATATAAATATTTcTGCATATAAATTGTAACTGATGTAAGAGGTTTCATATTGCTAATAGCAGCTACAATCCAGCTACCATTCTGCTTTTATTTTATGGTTGGGATAAGGCTGGATTATTCTGAGTCCAAGCTAGGCCCTTTTGCTAATCATGTTCATACCTCTTATCTTCCTCCCACAGCTCCTGGGCAACGTGCTGGTCTGTGTGCTGGCCCATCACTTTGGCAAAGCACGTGAGATCTGAATTCTGACACTATGAAGTGCCTTTTGTACTTAGCCTTTTTATTCATTGGGGTGAATTGCAAGTTCACCATAGTTTTTCCACACAACCAAAAAGGAAACTGGAAAAATGTTCCTTCTAATTACCATTATTGCCCGTCAAGCTCAGATTTAAATTGGCATAATGACTTAATAGGCACAGCCTTACAAGTCAAAATGCCCAAGAGTCACAAGGCTATTCAAGCAGACGGTTGGATGTGTCATGCTTCCAAATGGGTCACTACTTGTGATTTCCGCTGGTATGGACCGAAGTATATAACACATTCCATCCGATCCTTCACTCCATCTGTAGAACAATGCAAGGAAAGCATTGAACAAACGAAACAAGGAACTTGGCTGAATCCAGGCTTCCCTCCTCAAAGTTGTGGATATGCAACTGTGACGGATGCCGAAGCAGTGATTGTCCAGGTGACTCCTCACCATGTGCTGGTTGATGAATACACAGGAGAATGGGTTGATTCACAGTTCATCAACGGAAAATGCAGCAATTACATATGCCCCACTGTCCATAACTCTACAACCTGGCATTCTGACTATAAGGTCAAAGGGCTATGTGATTCTAACCTCATTTCCATGGACATCACCTTCTTCTCAGAGGACGGAGAGCTATCATCCCTGGGAAAGGAGGGCACAGGGTTCAGAAGTAACTACTTTGCTTATGAAACTGGAGGCAAGGCCTGCAAAATGCAATACTGCAAGCATTGGGGAGTCAGACTCCCATCAGGTGTCTGGTTCGAGATGGCTGATAAGGATCTCTTTGCTGCAGCCAGATTCCCTGAATGCCCAGAAGGGTCAAGTATCTCTGCTCCATCTCAGACCTCAGTGGATGTAAGTCTAATTCAGGACGTTGAGAGGATCTTGGATTATTCCCTCTGCCAAGAAACCTGGAGCAAAATCAGAGCGGGTCTTCCAATCTCTCCAGTGGATCTCAGCTATCTTGCTCCTAAAAACCCAGGAACCGGTCCTGCTTTCACCATAATCAATGGTACCCTAAAATACTTTGAGACCAGATACATCAGAGTCGATATTGCTGCTCCAATCCTCTCAAGAATGGTCGGAATGATCAGTGGAACTACCACAGAAAGGGAACTGTGGGATGACTGGGCACCATATGAAGACGTGGAAATTGGACCCAATGGAGTTCTGAGGACCAGTTCAGGATATAAGTTTCCTTTATACATGATTGGACATGGTATGTTGGACTCCGATCTTCATCTTAGCTCAAAGGCTCAGGTGTTCGAACATCCTCACATTCAAGACGCTGCTTCGCAACTTCCTGATGATGAGAGTTTATTTTTTGGTGATACTGGGCTATCCAAAPATCCAATCGAGCTTGTAGAAGGTTGGTTCAGTAGTTGGAAAAGCTCTATTGCCTCTTTTTTCTTTATCATAGGGTTAATCATTGGACTATTCTTGGTTCTCCGAGTTGGTATCCATCTTTGCATTAAATTAAAGCACACCAAGAAAAGACAGATTTATACAGACATAGAGATGAACCGACTTGGAAAGTAACTCAAATCCTGCACAACAGATTCTTCATGTTTGGACCAAATCAACTTGTGATACCATGCTCAAAGAGGCCTCAATTATATTTGAGTTTTTAATTTTTATGAAAAAAAAAAAAAAAAACGGAATTCACCCCACCAGTGCAGGCTGCCTATCAGAAAGTGGTGGCTGGTGTGGCTAATGCCCTGGCCCACAAGTATCACTAAGCTCGCTTTCTTGCTGTCCAATTTCTATTAAAGGTTCCTTTGTTCCCTAAGTCCAACTACTAAACTGGGGGATATTATGAAGGGCCTTGAGCATCTGGATTCTGCCTAATAAAAAACATTTATTTTCATTGCAATGATGTATTTAAATTATTTCTGAATATTTTACTAAAAAGGGAATGTGGGAGGTCAGTGCATTTAAAACATAAAGAAATGAAGAGCTAGTTCAAACCTTGGGAAAATACACTATATCTTAAACTCCATGAAAGAAGGTGAGGCTGCAAACAGCTAATGCACATTGGCAACAGCCCCTGATGCCTATGCCTTATTCATCCCTCAGAAAAGGATTCAAGTAGAGGCTTGATTTGGAGGTTAAAGTTTTGCTATGCTGTATTTTACATTACTTATTGTTTTAGCTGTCCTCATGAATGTCTTTTCACTACCCATTTGCTTATCCTGCATCTCTCAGCCTTGACTCCACTCAGTTCTCTTGCTTAGAGATACCACCTTTCCCCTGAAGTGTTCCTTCCATGTTTTACGGCGAGATGGTTTCTCCTCGCCTGGCCACTCAGCCTTAGTTGTCTCTGTTGTCTTATAGAGGTCTACTTGAAGAAGGAAAAACAGGGGGCATGGTTTGACTGTCCTGTGAGCCCTTCTTCCCTGCCTCCCCCACTCACAGTGACCCGGAATCCCTCGACATGGCAGTCTAGCACTAGTGCGGCCGCAGATCTGCTTCCTCGCTCACTGACTCGCTGCGCTCGGTCGTTCGGCTGCGGCGAGCGGTATCAGCTCACTCAAAGGCGGTAATACGGTTATCCACAGAATCAGGGGATAACGCAGGAAAGAACATGTGAGCAAAAGGCCAGCAAAAGGCCAGGAACCGTAAAAAGGCCGCGTTGCTGGCGTTTTTCCATAGGCTCCGCCCCCCTGACGAGCATCACAAAAATCGACGCTCAAGTCAGAGGTGGCGAAACCCGACAGGACTATAAAGATACCAGGCGTTTCCCCCTGGAAGCTCCCTCGTGCGCTCTCCTGTTCCGACCCTGCCGCTTACCGGATACCTGTCCGCCTTTCTCCCTTCGGGAAGCGTGGCGCTTTCTCATAGCTCACGCTGTAGGTATCTCAGTTCGGTGTAGGTCGTTCGCTCCAAGCTGGGCTGTGTGCACGAACCCCCCGTTCAGCCCGACCGCTGCGCCTTATCCGGTAACTATCGTCTTGAGTCCAACCCGGTAAGACACGACTTATCGCCACTGGCAGCAGCCACTGGTAACAGGATTAGCAGAGCGAGGTATGTAGGCGGTGCTACAGAGTTCTTGAAGTGGTGGCCTAACTACGGCTACACTAGAAGAACAGTATTTGGTATCTGCGCTCTGCTGAAGCCAGTTACCTTCGGAAAAAGAGTTGGTAGCTCTTGATCCGGCAAACAAACCACCGCTGGTAGCGGTGGTTTTTTTGTTTGCAAGCAGCAGATTACGCGCAGAAAAAAAGGATCTCAAGAAGATCCTTTGATCTTTTCTACGGGGTCTGACGCTCAGTGGAACGAAAACTCACGTTAAGGGATTTTGGTCATGAGATTATCAAAAAGGATCTTCACCTAGATCCTTTTAAATTAAAAATGAAGTTTTAAATCAATCTAAAGTATATATGAGTAAACTTGGTCTGACAGTTACCAATGCTTAATCAGTGAGGCACCTATCTCAGCGATCTGTCTATTTCGTTCATCCATAGTTGCCTGACTCCCCGTCGTGTAGATAACTACGATACGGGAGGGCTTACCATCTGGCCCCAGTGCTGCAATGATACCGCGAGACCCACGCTCACCGGCTCCAGATTTATCAGCAATAAACCAGCCAGCCGGAAGGGCCGAGCGCAGAAGTGGTCCTGCAACTTTATCCGCCTCCATCCAGTCTATTAATTGTTGCCGGGAAGCTAGAGTAAGTAGTTCGCCAGTTAATAGTTTGCGCAACGTTGTTGCCATTGCTACAGGCATCGTGGTGTCACGCTCGTCGTTTGGTATGGCTTCATTCAGCTCCGGTTCCCAACGATCAAGGCGAGTTACATGATCCCCCATGTTGTGCAAAAAAGCGGTTAGCTCCTTCGGTCCTCCGATCGTTGTCAGAAGTAAGTTGGCCGCAGTGTTATCACTCATGGTTATGGCAGCACTGCATAATTCTCTTACTGTCATGCCATCCGTAAGATGCTTTTCTGTGACTGGTGAGTACTCAACCAAGTCATTCTGAGAATAGTGTATGCGGCGACCGAGTTGCTCTTGCCCGGCGTCAATACGGGATAATACCGCGCCACATAGCAGAACTTTAAAAGTGCTCATCATTGGAAAACGTTCTTCGGGGCGAAAACTCTCAAGGATCTTACCGCTGTTGAGATCCAGTTCGATGTAACCCACTCGTGCACCCAACTGATCTTCAGCATCTTTTACTTTCACCAGCGTTTCTGGGTGAGCAAAAACAGGAAGGCAAAATGCCGCAAAAAAGGGAATAAGGGCGACACGGAAATGTTGAATACTCATACTCTTCCTTTTTCAATATTATTGAAGCATTTATCAGGGTTATTGTCTCATGAGCGGATACATATTTGAATGTATTTAGAAAAATAAACAAATAGGGGTTCCGCGCACATTTCCCCGAAAAGTGCCACCTGACGGGATCCCCTGAGGGGGCCCCCATGGGCTAGAGGATCCGGCCTCGGCCTCTGCATAAATAAAAAAAATTAGTCAGCCATGAGC

[1256] TABLE 28 Nucleotide sequence of plasmid pcDNA ™6.2/V5-DEST.GACGGATCGGGAGATCTCCCGATCCCCTATGGTGCACTCTCAGTACAATCTGCTCTGATGCCGCATAGTTAAGCCAGTATCTGCTCCCTGCTTGTGTGTTGGAGGTCGCTGAGTAGTGCGCGAGCAAAATTTAAGCTACAACAAGGCAAGGCTTGACCGACAATTGCATGAAGAATCTGCTTAGGGTTAGGCGTTTTGCGCTGCTTCGCGATGTACGGGCCAGATATACGCGTTGACATTGATTATTGACTAGTTATTAATAGTAATCAATTACGGGGTCATTAGTTCATAGCCCATATATGGAGTTCCGCGTTACATAACTTACGGTAAATGGCCCGCCTGGCTGACCGCCCAACGACCCCCGCCCATTGACGTCAATAATGACGTATGTTCCCATAGTAACGCCAATAGGGACTTTCCATTGACGTCAATGGGTGGAGTATTTACGGTAAACTGCCCACTTGGCAGTACATCAAGTGTATCATATGCCAAGTACGCCCCCTATTGACGTCAATGACGGTAAATGGCCCGCCTGGCATTATGCCCAGTACATGACCTTATGGGACTTTCCTACTTGGCAGTACATCTACGTATTAGTCATCGCTATTACCATGGTGATGCGGTTTTGGCAGTACATCAATGGGCGTGGATAGCGGTTTGACTCACGGGGATTTCCAAGTCTCCACCCCATTGACGTCAATGGGAGTTTGTTTTGGCACCAAAATCAACGGGACTTTCCAAAATGTCGTAACAACTCCGCCCCATTGACGCAAATGGGCGGTAGGCGTGTACGGTGGGAGGTCTATATAAGCAGAGCTCTCTGGCTAACTAGAGAACCCACTGCTTACTGGCTTATCGAAATTAATACGACTCACTATAGGGAGACCCAAGCTGGCTAGTTAAGCTATCAACAAGTTTGTACAAAAAAGCTGAACGAGAAACGTAAAATGATATAAATATCAATATATTAAATTAGATTTTGCATAAAAAACAGACTACATAATACTGTAAAACACAACATATCCAGTCACTATGATTCAACTACTTAGATGGTATTAGTGACCTGTAGTCGACCGACAGCCTTCCAAATGTTCTTCGGGTGATGCTGCCAACTTAGTCGACCGACAGCCTTCCAAATGTTCTTCTCAAACGGAATCGTCGTATCCAGCCTACTCGCTATTGTCCTCAATGCCGTATTAAATCATAAAAAGAAATAAGAAAAAGAGGTGCGAGCCTCTTTTTTGTGTGACAAAATAAAAACATCTACCTATTCATATACGCTAGTGTCATAGTCCTGAAAATCATCTGCATCAAGAACAATTTCACAACTCTTATACTTTTCTCTTACAAGTCGTTCGGCTTCATCTGGATTTTCAGCCTCTATACTTACTAAACGTGATAAAGTTTCTGTAATTTCTACTGTATCGACCTGCAGACTGGCTGTGTATAAGGGAGCCTGACATTTATATTCCCCAGAACATCAGGTTAATGGCGTTTTTGATGTCATTTTCGCGGTGGCTGAGATCAGCCACTTCTTCCCCGATAACGGAGACCGGCACACTGGCCATATCGGTGGTCATCATGCGCCAGCTTTCATCCCCGATATGCACCACCGGGTAAAGTTCACGGGAGACTTTATCTGACAGCAGACGTGCACTGGCCAGGGGGATCACCATCCGTCGCCCGGGCGTGTCAATAATATCACTCTGTACATCCACAAACAGACGATAACGGCTCTCTCTTTTATAGGTGTAAACCTTAAACTGCATTTCACCAGTCCCTGTTCTCGTCAGCAAAAGAGCCGTTCATTTCAATAAACCGGGCGACCTCAGCCATCCCTTCCTGATTTTCCGCTTTCCAGCGTTCGGCACGCAGACGACGGGCTTCATTCTGCATGGTTGTGCTTACCAGACCGGAGATATTGACATCATATATGCCTTGAGCAACTGATAGCTGTCGCTGTCAACTGTCACTGTAATACGCTGCTTCATAGCACACCTCTTTTTGACATACTTCGGGTATACATATCAGTATATATTCTTATACCGCAAAAATCAGCGCGCAAATACGCATACTGTTATCTGGCTTTTAGTAAGCCGGATCCACGCGATTACGCCCCGCCCTGCCACTCATCGCAGTACTGTTGTAATTCATTAAGCATTCTGCCGACATGGAAGCCATCACAGACGGCATGATGAACCTGAATCGCCAGCGGCATCAGCACCTTGTCGCCTTGCGTATAATATTTGCCCATGGTGAAAACGGGGGCGAAGAAGTTGTCCATATTGGCCACGTTTAAATCAAAACTGGTGAAACTCACCCAGGGATTGGCTGAGACGAAAAACATATTCTCAATAAACCCTTTAGGGAAATAGGCCAGGTTTTCACCGTAACACGCCACATCTTGCGAATATATGTGTAGAAACTGCCGGAAATCGTCGTGGTATTCACTCCAGAGCGATGAAAACGTTTCAGTTTGCTCATGGAAAACGGTGTAACAAGGGTGAACACTATCCCATATCACCAGCTCACCGTCTTTCATTGCCATACGGAATTCCGGATGAGCATTCATCAGGCGGGCAAGAATGTGAATAAAGGCCGGATAAAACTTGTGCTTATTTTTCTTTACGGTCTTTAAAAAGGCCGTAATATCCAGCTGAACGGTCTGGTTATAGGTACATTGAGCAACTGACTGAAATGCCTCAAAATGTTCTTTACGATGCCATTGGGATATATCAACGGTGGTATATCCAGTGATTTTTTTCTCCATTTTAGCTTCCTTAGCTCCTGAAAATCTCGATAACTCAAAAAATACGCCCGGTAGTGATCTTATTTCATTATGCTGAAAGTTGGAACCTCTTACGTGCCGATCAACGTCTCATTTTCGCCAAAAGTTGGCCCAGGGCTTCCCGGTATCAACAGGGACACCAGGATTTATTTATTCTGCGAAGTGATCTTCCGTCACAGGTATTTATTCGGCGCAAAGTGCGTCGGGTGATGCTGCCAACTTAGTCGACTACAGGTCACTAATACCATCTAAGTAGTTGATTCATAGTGACTGGATATGTTGTGTTTTACAGTATTATGTAGTCTGTTTTTTATGCAAAATCTAATTTAATATATTGATATTTATATCATTTTACGTTTCTCGTTCAGCTTTCTTGTACAAAGTGGTTGATCTAGAGGGCCCGCGGTTCGAAGGTAAGCCTATCCCTAACCCTCTCCTCGGTCTCGATTCTACGCGTACCGGTTAGTAATGAGTTTAAACGGGGGAGGCTAACTGAAACACGGAAGGAGACAATACCGGAAGGAACCCGCGCTATGACGGCAATAAAAAGACAGAATAAAACGCACGGGTGTTGGGTCGTTTGTTCATAAACGCGGGGTTCGGTCCCAGGGCTGGCACTCTGTCGATACCCCACCGAGACCCCATTGGGGCCAATACGCCCGCGTTTCTTCCTTTTCCCCACCCCACCCCCCAAGTTCGGGTGAAGGCCCAGGGCTCGCAGCCAACGTCGGGGCGGCAGGCCCTGCCATAGCAGATCTGCGCAGCTGGGGCTCTAGGGGGTATCCCCACGCGCCCTGTAGCGGCGCATTAAGCGCGGCGGGTGTGGTGGTTACGCGCAGCGTGAcCGCTACACTTGCCAGCGCCCTAGCGCCCGCTCCTTTCGCTTTCTTCCCTTCCTTTCTCGCCACGTTCGCCGGCTTTCCCCGTCAAGCTCTAAATCGGGGCATCCCTTTAGGGTTCCGATTTAGTGCTTTACGGCACCTCGACCCCAAAAAACTTGATTAGGGTGATGGTTCACGTAGTGGGCCATCGCCCTGATAGACGGTTTTTCGCCCTTTGACGTTGGAGTCCACGTTCTTTAATAGTGGACTCTTGTTCCAAACTGGAACAACACTCAACCCTATCTCGGTCTATTCTTTTGATTTATAAGGGATTTTGGGGATTTCGGCCTATTGGTTAAAAAATGAGCTGATTTAACAAAAATTTAACGCGAATTAATTCTGTGGAATGTGTGTCAGTTAGGGTGTGGAAAGTCCCCAGGCTCCCCAGCAGGCAGAAGTATGCAAAGCATGCATCTCAATTAGTCAGCAACCAGGTGTGGAAAGTCCCCAGGCTCCCCAGCAGGCAGAAGTATGCAAAGCATGCATCTCAATTAGTCAGCAACCATAGTCCCGCCCCTAACTCCGCCCATCCCGCCCCTAACTCCGCCCAGTTCCGCCCATTCTCCGCCCCATGGCTGACTAATTTTTTTTATTTATGCAGAGGCCGAGGCCGCCTCTGCCTCTGAGCTATTCCAGAAGTAGTGAGGAGGCTTTTTTGGAGGCCTAGGCTTTTGCAAAAAGCTCCCGGGAGCTTGTATATCCATTTTCGGATCTGATCAGCACGTGTTGACAATTAATCATCGGCATAGTATATCGGCATAGTATAATACGACAAGGTGAGGAACTAAACCATGGCCAAGCCTTTGTCTCAAGAAGAATCCACCCTCATTGAAAGAGCAACGGCTACAATCAACAGCATCCCCATCTCTGAAGACTACAGCGTCGCCAGCGCAGCTCTCTCTAGCGACGGCCGCATCTTCACTGGTGTCAATGTATATCATTTTACTGGGGGACCTTGTGCAGAACTCGTGGTGCTGGGCACTGCTGCTGCTGCGGCAGCTGGCAACCTGACTTGTATCGTCGCGATCGGAAATGAGAACAGGGGCATCTTGAGCCCCTGCGGACGGTGCCGACAGGTGCTTCTCGATCTGCATCCTGGGATCAAAGCCATAGTGAAGGACAGTGATGGACAGCCGACGGCAGTTGGGATTCGTGAATTGCTGCCCTCTGGTTATGTGTGGGAGGGCTAAGCACTTCGTGGCCGAGGAGCAGGACTGACACGTGCTACGAGATTTCGATTCCACCGCCGCCTTCTATGAAAGGTTGGGCTTCGGAATCGTTTTCCGGGACGCCGGCTGGATGATCCTCCAGCGCGGGGATCTCATGCTGGAGTTCTTCGCCCACCCCAACTTGTTTATTGCAGCTTATAATGGTTACAAATAAAGCAATAGCATCACAAATTTCACAAATAAAGCATTTTTTTCACTGCATTCTAGTTGTGGTTTGTCCAAACTCATCAATGTATCTTATCATGTCTGTATACCGTCGACCTCTAGCTAGAGCTTGGCGTAATCATGGTCATAGCTGTTTCCTGTGTGAAATTGTTATCCGCTCACAATTCCACACAACATACGAGCCGGAAGCATAAAGTGTAAAGCCTGGGGTGCCTAATGAGTGAGCTAACTCACATTAATTGCGTTGCGCTCACTGCCCGCTTTCCAGTCGGGAAACCTGTCGTGCCAGCTGCATTAATGAATCGGCCAACGCGCGGGGAGAGGCGGTTTGCGTATTGGGCGCTCTTCCGCTTCCTCGCTCACTGACTCGCTGCGCTCGGTCGTTCGGCTGCGGCGAGCGGTATCAGCTCACTCAAAGGCGGTAATACGGTTATCCACAGAATCAGGGGATAACGCAGGAAAGAACATGTGAGCAAAAGGCCAGCAAAAGGCCAGGAACCGTAAAAAGGCCGCGTTGCTGGCGTTTTTCCATAGGCTCCGCCCCCCTGACGAGCATCACAAAAATCGACGCTCAAGTCAGAGGTGGCGAAACCCGACAGGACTATAAAGATACCAGGCGTTTCCCCCTGGAAGCTCCCTCGTGCGCTCTCCTGTTCCGACCCTGCCGCTTACCGGATACCTGTCCGCCTTTCTCCCTTCGGGAAGCGTGGCGCTTTCTCATAGCTCACGCTGTAGGTATCTCAGTTCGGTGTAGGTCGTTCGCTCCAAGCTGGGCTGTGTGCACGAACCCCCCGTTCAGCCCGACCGCTGCGCCTTATCCGGTAACTATCGTCTTGAGTCCAACCCGGTAAGACACGACTTATCGCCACTGGCAGCAGCCACTGGTAACAGGATTAGCAGAGCGAGGTATGTAGGCGGTGCTACAGAGTTCTTGAAGTGGTGGCCTAACTACGGCTACACTAGAAGAACAGTATTTGGTATCTGCGCTCTGCTGAAGCCAGTTACCTTCGGAAAAAGAGTTGGTAGCTCTTGATCCGGCAAACAAACCACCGCTGGTAGCGGTTTTTTTGTTTGCAAGCAGCAGATTACGCGCAGAAAAAAAGGATCTCAAGAAGATCCTTTGATCTTTTCTACGGGGTCTGACGCTCAGTGGAACGAAAACTCACGTTAAGGGATTTTGGTCATGAGATTATCAAAAAGGATCTTCACCTAGATCCTTTTAAATTAAAAATGAAGTTTTAAATCAATCTAAAGTATATATGAGTAAACTTGGTCTGACAGTTACCAATGCTTAATCAGTGAGGCACCTATCTCAGCGATCTGTCTATTTCGTTCATCCATAGTTGCCTGACTCCCCGTCGTGTAGATAACTACGATACGGGAGGGCTTACCATCTGGCCCCAGTGCTGCAATGATACCGCGAGACCCACGCTCACCGGCTCCAGATTTATCAGCAATAAACCAGCCAGCCGGAAGGGCCGAGCGCAGAAGTGGTCCTGCAACTTTATCCGCCTCCATCCAGTCTATTAATTGTTGCCGGGAAGCTAGAGTAAGTAGTTCGCCAGTTAATAGTTTGCGCAACGTTGTTGCCATTGCTACAGGCATCGTGGTGTCACGCTCGTCGTTTGGTATGGCTTCATTCAGCTCCGGTTCCCAACGATCAAGGCGAGTTACATGATCCCCCATGTTGTGCAAAAAAGCGGTTAGCTCCTTCGGTCCTCCGATCGTTGTCAGAAGTAAGTTGGCCGCAGTGTTATCACTCATGGTTATGGCAGCACTGCATAATTCTCTTACTGTCATGCCATCCGTAAGATGCTTTTCTGTGACTGGTGAGTACTCAACCAAGTCATTCTGAGAATAGTGTATGCGGCGACCGAGTTGCTCTTGCCCGGCGTCAATACGGGATAATACCGCGCCACATAGCAGAACTTTAAAAGTGCTCATCATTGGAAAACGTTCTTCGGGGCGAAAACTCTCAAGGATCTTACCGCTGTTGAGATCCAGTTCGATGTAACCCACTCGTGCACCCAACTGATCTTCAGCATCTTTTACTTTCACCAGCGTTTCTGGGTGAGCAAAAACAGGAAGGCAAAATGCCGCAAAAAAGGGAATAAGGGCGACACGGAAATGTTGAATACTCATACTCTTCCTTTTTCAATATTATTGAAGCATTTATCAGGGTTATTGTCTCATGAGCGGATACATATTTGAATGTATTTAGAAAAATAAACAAATAGGGGTTCCGCGCACATTTCCCCGAAAAGTGCCACCTGACGTC

[1257] TABLE 29 Nucleotide sequence of plasmid pcDNA ™6.2/GFP-DEST.GACGGATCGGGAGATCTCCCGATCCCCTATGGTGCACTCTCAGTACAATCTGCTCTGATGCCGCATAGTTAAGCCAGTATCTGCTCCCTGCTTGTGTGTTGGAGGTCGCTGAGTAGTGCGCGAGCAAAATTTAAGCTACAACAAGGCAAGGCTTGACCGACAATTGCATGAAGAATCTGCTTAGGGTTAGGCGTTTTGCGCTGCTTCGCGATGTACGGGCCAGATATACGCGTTGACATTGATTATTGACTAGTTATTAATAGTAATCAATTACGGGGTCATTAGTTCATAGCCCATATATGGAGTTCCGCGTTACATAACTTACGGTAAATGGCCCGCCTGGCTGACCGCCCAACGACCCCCGCCCATTGACGTCAATAATGACGTATGTTCCCATAGTAACGCCAATAGGGACTTTCCATTGACGTCAATGGGTGGAGTATTTACGGTAAACTGCCCACTTGGCAGTACATCAAGTGTATCATATGCCAAGTACGCCCCCTATTGACGTCAATGACGGTAAATGGCCCGCCTGGCATTATGCCCAGTACATGACCTTATGGGACTTTCCTACTTGGCAGTACATCTACGTATTAGTCATCGCTATTACCATGGTGATGCGGTTTTGGCAGTACATCAATGGGCGTGGATAGCGGTTTGACTCACGGGGATTTCCAAGTCTCCACCCCATTGACGTCAATGGGAGTTTGTTTTGGCACCAAAATCAACGGGACTTTCCAAAATGTCGTAACAACTCCGCCCCATTGACGCAAATGGGCGGTAGGCGTGTACGGTGGGAGGTCTATATAAGCAGAGCTCTCTGGCTAACTAGAGAACCCACTGCTTACTGGCTTATCGAAATTAATACGACTCACTATAGGGAGACCCAAGCTGGCTAGTTAAGCTATCAACAAGTTTGTACAAAAAAGCTGAACGAGAAACGTAAAATGATATAAATATCAATATATTAAATTAGATTTTGCATAAAAAACAGACTACATAATACTGTAAAACACAACATATCCAGTCACTATGAATCAACTACTTAGATGGTATTAGTGACCTGTAGTCGACCGACAGCCTTCCAAATGTTCTTCGGGTGATGCTGCCAACTTAGTCGACCGACAGCCTTCCAAATGTTCTTCTCAAACGGAATCGTCGTATCCAGCCTACTCGCTATTGTCCTCAATGCCGTATTAAATCATAAAAAGAAATAAGAAAAAGAGGTGCGAGCCTCTTTTTTGTGTGACAAAATAAAAACATCTACCTATTCATATACGCTAGTGTCATAGTCCTGAAAATCATCTGCATCAAGAACAATTTCACAACTCTTATACTTTTCTCTTACAAGTCGTTCGGCTTCATCTGGATTTTCAGCCTCTATACTTACTAAACGTGATAAAGTTTCTGTAATTTCTACTGTATCGACCTGCAGACTGGCTGTGTATAAGGGAGCCTGACATTTATATTCCCCAGAACATCAGGTTAATGGCGTTTTTGATGTCATTTTCGCGGTGGCTGAGATCAGCCACTTCTTCCCCGATAACGGAGACCGGCACACTGGCCATATCGGTGGTCATCATGCGCCAGCTTTCATCCCCGATATGCACCACCGGGTAAAGTTCACGGGAGACTTTATCTGACAGCAGACGTGCACTGGCCAGGGGGATCACCATCCGTCGCCCGGGCGTGTCAATAATATCACTCTGTACATCCACAAACAGACGATAACGGCTCTCTCTTTTATAGGTGTAAACCTTAAACTGCATTTCACCAGTCCCTGTTCTCGTCAGCAAAAGAGCCGTTCATTTCAATAAACCGGGCGACCTCAGCCATCCCTTCCTGATTTTCCGCTTTCCAGCGTTCGGCACGCAGACGACGGGCTTCATTCTGCATGGTTGTGCTTACCAGACCGGAGATATTGACATCATATATGCCTTGAGCAACTGATAGCTGTCGCTGTCAACTGTCACTGTAATACGCTGCTTCATAGCACACCTCTTTTTGACATACTTCGGGTATACATATCAGTATATATTCTTATACCGCAAAAATCAGCGCGCAAATACGCATACTGTTATCTGGCTTTTAGTAGGCCGGATCCACGCGATTACGCCCCGCCCTGCCACTCATCGCAGTACTGTTGTAATTCATTAAGCATTCTGCCGACATGGAAGCCATCACAGACGGCATGATGAACCTGAATCGCCAGCGGCATCAGCACCTTGTCGCCTTGCGTATAATATTTGCCCATGGTGAAAACGGGGGCGAAGAAGTTGTCCATATTGGCCACGTTTAAATCAAAACTGGTGAAACTCACCCAGGGATTGGCTGAGACGAAAAACATATTCTCAATAAACCCTTTAGGGAAATAGGCCAGGTTTTCACCGTAACACGCCACATCTTGCGAATATATGTGTAGAAACTGCCGGAAATCGTCGTGGTATTCACTCCAGAGCGATGAAAACGTTTCAGTTTGCTCATGGAAAACGGTGTAACAAGGGTGAACACTATCCCATATCACCAGCTCACCGTCTTTCATTGCCATACGGAATTCCGGATGAGCATTCATCAGGCGGGCAAGAATGTGAATAAAGGCCGGATAAAACTTGTGCTTATTTTTCTTTACGGTCTTTAAAAAGGCCGTAATATCCAGCTGAACGGTCTGGTTATAGGTACATTGAGCAACTGACTGAAATGCCTCAAAATGTTCTTTACGATGCCATTGGGATATATCAACGGTGGTATATCCAGTGATTTTTTTCTCCATTTTAGCTTCCTTAGCTCCTGAAAATCTCGATAACTCAAAAAATACGCCCGGTAGTGATCTTATTTCATTATGGTGAAAGTTGGAACCTCTTACGTGCCGATCAACGTCTCATTTTCGCCAAAAGTTGGCCCAGGGCTTCCCGGTATCAACAGGGACACCAGGATTTATTTATTCTGCGAAGTGATCTTCCGTCACAGGTATTTATTCGGCGCAAAGTGCGTCGGGTGATGCTGCCAACTTAGTCGACTACAGGTCACTAATACCATCTAAGTAGTTGATTCATAGTGACTGGATATGTTGTGTTTTACAGTATTATGTAGTCTGTTTTTTATGCAAAATCTAATTTAATATATTGATATTTATATCATTTTACGTTTCTCGTTCAGCTTTCTTGTACAAAGTGGTTGATCTAGAGGGCCCCGCGGCTAGCAAAGGAGAAGAACTTTTCACTGGAGTTGTCCCAATTCTTGTTGAATTAGATGGTGATGTTAATGGGCACAAATTTTCTGTCAGTGGAGAGGGTGAAGGTGATGCTACATACGGAAAGCTTACCCTTAAATTTATTTGCACTACTGGAAAACTACCTGTTCCATGGCCAACACTTGTCACTACTTTCTCTTATGGTGTTCAATGCTTTTCCCGTTATCCGGATCATATGAAACGGCATGACTTTTTCAAGAGTGCCATGCCCGAAGGTTATGTACAGGAACGCACTATATCTTTCAAAGATGACGGGAACTACAAGACGCGTGCTGAAGTCAAGTTTGAAGGTGATACCCTTGTTAATCGTATCGAGTTAAAAGGTATTGATTTTAAAGAAGATGGAAACATTCTCGGACACAAACTCGAGTACAACTATAACTCACACAATGTATACATCACGGCAGACAAACAAAAGAATGGAATCAAAGCTAACTTCAAAATTCGTCACAACATTGAAGATGGATCCGTTCAACTAGCAGACCATTATCAACAAAATACTCCAATTGGCGATGGCCCTGTCCTTTTACCAGACAACCATTACCTGTCGACACAATCTGCCCTTTCGAAAGATCCCAACGAAAAGCGTGACCACATGGTCCTTCTTGAGTTTGTAACTGCTGCTGGGATTACACATGGCATGGATGAATAGTAATGAGTCCACGTTTAAACGGGGGAGGCTAACTGAAACACGGAAGGAGACAATACCGGAAGGAACCCGCGCTATGACGGCAATAAAAAGACAGAATAAAACGCACGGGTGTTGGGTCGTTTGTTCATAAACGCGGGGTTCGGTCCCAGGGCTGGCACTCTGTCGATACCCCACCGAGACCCCATTGGGGCCAATACGCCCGCGTTTCTTCCTTTTCCCCACCCCACCCCCCAAGTTCGGGTGAAGGCCCAGGGCTCGCAGCCAACGTCGGGGCGGCAGGCCCTGCCATAGCAGATCTGCGCAGCTGGGGCTCTAGGGGGTATCCCCACGCGCCCTGTAGCGGCGCATTAAGCGCGGCGGGTGTGGTGGTTACGCGCAGCGTGACCGCTACACTTGCCAGCGCCCTAGCGCCCGCTCCTTTCGCTTTCTTCCCTTCCTTTCTCGCCACGTTCGCCGGCTTTCCCCGTCAAGCTCTAAATCGGGGCATCCCTTTAGGGTTCCGATTTAGTGCTTTACGGCACCTCGACCCCAAAAAACTTGATTAGGGTGATGGTTCACGTAGTGGGCCATCGCCCTGATAGACGGTTTTTCGCCCTTTGACGTTGGAGTCCACGTTCTTTAATAGTGGACTCTTGTTCCAAACTGGAACAACACTCAACCCTATCTCGGTCTATTCTTTTGATTTATAAGGGATTTTGGGGATTTCGGCCTATTGGTTAAAAAATGAGCTGATTTAACAAAAATTTAACGCGAATTAATTCTGTGGAATGTGTGTCAGTTAGGGTGTGGAAAGTCCCCAGGCTCCCCAGCAGGCAGAAGTATGCAAAGCATGCATCTCAATTAGTCAGCAACCAGGTGTGGAAAGTCCCCAGGCTCCCCAGCAGGCAGAAGTATGCAAAGCATGCATCTCAATTAGTCAGCAACCATAGTCCCGCCCCTAACTCCGCCCATCCCGCCCCTAACTCCGCCCAGTTCCGCCCATTCTCCGCCCCATGGCTGACTAATTTTTTTTATTTATGCAGAGGCCGAGGCCGCCTCTGCCTCTGAGCTATTCCAGAAGTAGTGAGGAGGCTTTTTTGGAGGCCTAGGCTTTTGCAAAAAGCTCCCGGGAGCTTGTATATCCATTTTCGGATCTGATCAGCACGTGTTGACAATTAATCATCGGCATAGTATATCGGCATAGTATAATACGACAAGGTGAGGAACTAAACCATGGCCAAGCCTTTGTCTCAAGAAGAATCCACCCTCATTGAAAGAGCAACGGCTACAATCAACAGCATCCCCATCTCTGAAGACTACAGCGTCGCCAGCGCAGCTCTCTCTAGCGACGGCCGCATCTTCACTGGTGTCAATGTATATCATTTTACTGGGGGACCTTGTGCAGAACTCGTGGTGCTGGGCACTGCTGCTGCTGCGGCAGCTGGCAACCTGACTTGTATCGTCGCGATCGGAAATGAGAACAGGGGCATCTTGAGCCCCTGCGGACGGTGCCGACAGGTGCTTCTCGATCTGCATCCTGGGATCAAAGCCATAGTGAAGGACAGTGATGGACAGCCGACGGCAGTTGGGATTCGTGAATTGCTGCCCTCTGGTTATGTGTGGGAGGGCTAAGCACTTCGTGGCCGAGGAGCAGGACTGACACGTGCTACGAGATTTCGATTCCACCGCCGCCTTCTATGAAAGGTTGGGCTTCGGAATCGTTTTCCGGGACGCCGGCTGGATGATCCTCCAGCGCGGGGATCTCATGCTGGAGTTCTTCGCCCACCCCAACTTGTTTATTGCAGCTTATAATGGTTACAAATAAAGCAATAGCATCACAAATTTCACAAATAAAGCATTTTTTTCACTGCATTCTAGTTGTGGTTTGTCCAAACTCATCAATGTATCTTATCATGTCTGTATACCGTCGACCTCTAGCTAGAGCTTGGCGTAATCATGGTCATAGCTGTTTCCTGTGTGAAATTGTTATCCGCTCACAATTCCACACAACATACGAGCCGGAAGCATAAAGTGTAAAGCCTGGGGTGCCTAATGAGTGAGCTAACTCACATTAATTGCGTTGCGCTCACTGCCCGCTTTCCAGTCGGGAAACCTGTCGTGCCAGCTGCATTAATGAATCGGCCAACGCGCGGGGAGAGGCGGTTTGCGTATTGGGCGCTCTTCCGCTTCCTCGCTCACTGACTCGCTGCGCTCGGTCGTTCGGCTGCGGCGAGCGGTATCAGCTCACTCAAAGGCGGTAATACGGTTATCCACAGAATCAGGGGATAACGCAGGAAAGAACATGTGAGCAAAAGGCCAGCAAAAGGCCAGGAACCGTAAAAAGGCCGCGTTGCTGGCGTTTTTCCATAGGCTCCGCCCCCCTGACGAGCATCACAAAAATCGACGCTCAAGTCAGAGGTGGCGAAACCCGACAGGACTATAAAGATACCAGGCGTTTCCCCCTGGAAGCTCCCTCGTGCGCTCTCCTGTTCCGACCCTGCCGCTTACCGGATACCTGTCCGCCTTTCTCCCTTCGGGAAGCGTGGCGCTTTCTCATAGCTCACGCTGTAGGTATCTCAGTTCGGTGTAGGTCGTTCGCTCCAAGCTGGGCTGTGTGCACGAACCCCCCGTTCAGCCCGACCGCTGCGCCTTATCCGGTAACTATCGTCTTGAGTCCAACCCGGTAAGACACGACTTATCGCCACTGGCAGCAGCCACTGGTAACAGGATTAGCAGAGCGAGGTATGTAGGCGGTGCTACAGAGTTCTTGAAGTGGTGGCCTAACTACGGCTACACTAGAAGAACAGTATTTGGTATCTGCGCTCTGCTGAAGCCAGTTACCTTCGGAAAAAGAGTTGGTAGCTCTTGATCCGGCAAACAAACCACCGCTGGTAGCGGTTTTTTTGTTTGCAAGCAGCAGATTACGCGCAGAAAAAAAGGATCTCAAGAAGATCCTTTGATCTTTTCTACGGGGTCTGACGCTCAGTGGAACGAAAACTCACGTTAAGGGATTTTGGTCATGAGATTATCAAAAAGGATCTTCACCTAGATCCTTTTAAATTAAAAATGAAGTTTTAAATCAATCTAAAGTATATATGAGTAAACTTGGTCTGACAGTTACCAATGCTTAATCAGTGAGGCACCTATCTCAGCGATCTGTCTATTTCGTTCATCCATAGTTGCCTGACTCCCCGTCGTGTAGATAACTACGATACGGGAGGGCTTACCATCTGGCCCCAGTGCTGCAATGATACCGCGAGACCCACGCTCACCGGCTCCAGATTTATCAGCAATAAACCAGCCAGCCGGAAGGGCCGAGCGCAGAAGTGGTCCTGCAACTTTATCCGCCTCCATCCAGTCTATTAATTGTTGCCGGGAAGCTAGAGTAAGTAGTTCGCCAGTTAATAGTTTGCGCAACGTTGTTGCCATTGCTACAGGCATCGTGGTGTCACGCTCGTCGTTTGGTATGGCTTCATTCAGCTCCGGTTCCCAACGATCAAGGCGAGTTACATGATCCCCCATGTTGTGCAAAAAAGCGGTTAGCTCCTTCGGTCCTCCGATCGTTGTCAGAAGTAAGTTGGCCGCAGTGTTATCACTCATGGTTATGGCAGCACTGCATAATTCTCTTACTGTCATGCCATCCGTAAGATGCTTTTCTGTGACTGGTGAGTACTCAACCAAGTCATTCTGAGAATAGTGTATGCGGCGACCGAGTTGCTCTTGCCCGGCGTCAATACGGGATAATACCGCGCCACATAGCAGAACTTTAAAAGTGCTCATCATTGGAAAACGTTCTTCGGGGCGAAAACTCTCAAGGATCTTACCGCTGTTGAGATCCAGTTCGATGTAACCCACTCGTGCACCCAACTGATCTTCAGCATCTTTTACTTTCACCAGCGTTTCTGGGTGAGCAAAAACAGGAAGGCAAAATGCCGCAAAAAAGGGAATAAGGGCGACACGGAAATGTTGAATACTCATACTCTTCCTTTTTCAATATTATTGAAGCATTTATCAGGGTTATTGTCTCATGAGCGGATACATATTTGAATGTATTTAGAAAAATAAACAAATAGGGGTTCCGCGCACATTTCCCCGAAAAGTGCCACCTGACGTC

[1258] TABLE 30 Amino acid sequence of a polypeptide having β-lactamaseactivity. Met Gly His Pro Glu Thr Leu Val Lys Val Lys Asp Ala Glu AspGln   1               5                  10                  15 Leu GlyAla Arg Val Gly Tyr Ile Glu Leu Asp Leu Asn Ser Gly Lys             20                  25                  30 Ile Leu Glu SerPhe Arg Pro Glu Glu Arg Phe Pro Met Met Ser Thr         35                  40                  45 Phe Lys Val Leu LeuCys Gly Ala Val Leu Ser Arg Asp Asp Ala Gly     50                  55                  60 Gln Glu Gln Leu Gly ArgArg Ile His Tyr Ser Gln Asn Asp Leu Val 65                  70                  75                  80 Glu TyrSer Pro Val Thr Glu Lys His Leu Thr Asp Gly Met Thr Val                 85                  90                  95 Arg Glu LeuCys Ser Ala Ala Ile Thr Met Ser Asp Asn Thr Ala Ala            100                 105                 110 Asn Leu Leu LeuThr Thr Ile Gly Gly Pro Lys Glu Leu Thr Ala Phe        115                 120                 125 Leu His Asn Met GlyAsp His Val Thr Arg Leu Asp His Trp Glu Pro    130                 135                 140 Glu Leu Asn Glu Ala IlePro Asn Asp Glu Arg Asp Thr Thr Met Pro145                 150                 155                 160 Val AlaMet Ala Thr Thr Leu Arg Lys Leu Leu Thr Gly Glu Leu Leu                165                 170                 175 Thr Leu AlaSer Arg Gln Gln Leu Ile Asp Trp Met Glu Ala Asp Lys            180                 185                 190 Val Ala Gly ProLeu Leu Arg Ser Ala Leu Pro Ala Gly Trp Phe Ile        195                 200                 205 Ala Asp Lys Ser GlyAla Gly Glu Arg Gly Ser Arg Gly Ile Ile Ala    210                 215                 220 Ala Leu Gly Pro Asp GlyLys Pro Ser Arg Ile Val Val Ile Tyr Thr225                 230                 235                 240 Thr GlySer Gln Ala Thr Met Asp Glu Arg Asn Arg Gln Ile Ala Glu                245                 250                 255 Ile Gly AlaSer Leu Ile Lys His Trp     260         265

[1259] TABLE 31 Nucleotide sequence of pLenti4TO/V5-DEST.aatgtagtcttatgcaatactcttgtagtcttgcaacatggtaacgatgagttagcaacatgccttacaaggagagaaaaagcaccgtgcatgccgattggtggaagtaaggtggtacgatcgtgccttattaggaaggcaacagacgggtctgacatggattggacgaaccactgaattgccgcattgcagagatattgtatttaagtgcctagctcgatacataaacgggtctctctggttagaccagatctgagcctgggagctctctggctaactagggaacccactgcttaagcctcaataaagcttgccttgagtgcttcaagtagtgtgtgcccgtctgttgtgtgactctggtaactagagatccctcagacccttttagtcagtgtggaaaatctctagcagtggcgcccgaacagggacttgaaagcgaaagggaaaccagaggagctctctcgacgcaggactcggcttgctgaagcgcgcacggcaagaggcgaggggcggcgactggtgagtacgccaaaaattttgactagcggaggctagaaggagagagatgggtgcgagagcgtcagtattaagcgggggagaattagatcgcgatgggaaaaaattcggttaaggccagggggaaagaaaaaatataaattaaaacatatagtatgggcaagcagggagctagaacgattcgcagttaatcctggcctgttagaaacatcagaaggctgtagacaaatactgggacagctacaaccatcccttcagacaggatcagaagaacttagatcattatataatacagtagcaaccctctattgtgtgcatcaaaggatagagataaaagacaccaaggaagctttagacaagatagaggaagagcaaaacaaaagtaagaccaccgcacagcaagcggccgctgatcttcagacctggaggaggagatatgagggacaattggagaagtgaattatataaatataaagtagtaaaaattgaaccattaggagtagcacccaccaaggcaaagagaagagtggtgcagagagaaaaaagagcagtgggaataggagctttgttccttgggttcttgggagcagcaggaagcactatgggcgcagcgtcaatgacgctgacggtacaggccagacaattattgtctggtatagtgcagcagcagaacaatttgctgagggctattgaggcgcaacagcatctgttgcaactcacagtctggggcatcaagcagctccaggcaagaatcctggctgtggaaagatacctaaaggatcaacagctcctggggatttggggttgctctggaaaactcatttgcaccactgctgtgccttggaatgctagttggagtaataaatctctggaacagatttggaatcacacgacctggatggagtgggacagagaaattaacaattacacaagcttaatacactccttaattgaagaatcgcaaaaccagcaagaaaagaatgaacaagaattattggaattagataaatgggcaagtttgtggaattggtttaacataacaaattggctgtggtatataaaattattcataatgatagtaggaggcttggtaggtttaagaatagtttttgctgtactttctatagtgaatagagttaggcagggatattcaccattatcgtttcagacccacctcccaaccccgaggggacccgacaggcccgaaggaatagaagaagaaggtggagagagagacagagacagatccattcgattagtgaacggatctcgacggtatcgataagcttgggagttccgcgttacataacttacggtaaatggcccgcctggctgaccgcccaacgacccccgcccattgacgtcaataatgacgtatgttcccatagtaacgccaatagggactttccattgacgtcaatgggtggagtatttacggtaaactgcccacttggcagtacatcaagtgtatcatatgccaagtacgccccctattgacgtcaatgacggtaaatggcccgcctggcattatgcccagtacatgaccttatgggactttcctacttggcagtacatctacgtattagtcatcgctattaccatggtgatgcggttttggcagtacatcaatgggcgtggatagcggtttgactcacggggatttccaagtctccaccccattgacgtcaatgggagtttgttttggcaccaaaatcaacgggactttccaaaatgtcgtaacaactccgccccattgacgcaaatgggcggtaggcgtgtacggtgggaggtctatataagcagagctctccctatcagtgatagagatctccctatcagtgatagagatcgtcgactagtccagtgtggtggaattctgcagatatcaacaagtttgtacaaaaaagctgaacgagaaacgtaaaatgatataaatatcaatatattaaattagattttgcataaaaaacagactacataatactgtaaaacacaacatatccagtcactatggcggccgcattaggcaccccaggctttacactttatgcttccggctcgtataatgtgtggattttgagttaggatccggcgagattttcaggagctaaggaagctaaaatggagaaaaaaatcactggatataccaccgttgatatatcccaatggcatcgtaaagaacattttgaggcatttcagtcagttgctcaatgtacctataaccagaccgttcagctggatattacggcctttttaaagaccgtaaagaaaaataagcacaagttttatccggcctttattcacattcttgcccgcctgatgaatgctcatccggaattccgtatggcaatgaaagacggtgagctggtgatatgggatagtgttcacccttgttacaccgttttccatgagcaaactgaaacgttttcatcgctctggagtgaataccacgacgatttccggcagtttctacacatatattcgcaagatgtggcgtgttacggtgaaaacctggcctatttccctaaagggtttattgagaatatgtttttcgtctcagccaatccctgggtgagtttcaccagttttgatttaaacgtggccaatatggacaacttcttcgcccccgttttcaccatgggcaaatattatacgcaaggcgacaaggtgctgatgccgctggcgattcaggttcatcatgccgtctgtgatggcttccatgtcggcagaatgcttaatgaattacaacagtactgcgatgagtggcagggcggggcgtaaagatctggatccggcttactaaaagccagataacagtatgcgtatttgcgcgctgatttttgcggtataagaatatatactgatatgtatacccgaagtatgtcaaaaagaggtgtgctatgaagcagcgtattacagtgacagttgacagcgacagctatcagttgctcaaggcatatatgatgtcaatatctccggtctggtaagcacaaccatgcagaatgaagcccgtcgtctgcgtgccgaacgctggaaagcggaaaatcaggaagggatggctgaggtcgcccggtttattgaaatgaacggctcttttgctgacgagaacagggactggtgaaatgcagtttaaggtttacacctataaaagagagagccgttatcgtctgtttgtggatgtacagagtgatattattgacacgcccgggcgacggatggtgatccccctggccagtgcacgtctgctgtcagataaagtctcccgtgaactttacccggtggtgcatatcggggatgaaagctggcycatgatgaccaccgatatggccagtgtgccggtctccgttatcggggaagaagtggctgatctcagccaccgcgaaaatgacatcaaaaacgccattaacctgatgttctggggaatataaatgtcaggctccgttatacacagccagtctgcaggtcgaccatagtgactggatatgttgtgttttacagtattatgtagtctgttttttatgcaaaatctaatttaatatattgatatttatatcattttacgtttctcgttcagctttcttgtacaaagtggttgatatccagcacagtggcggccgctcgagtctagagggcccgcggttcgaaggtaagcctatccctaaccctctcctcggtctcgattctacgcgtaccggttagtaatgagtttggaattaattctgtggaatgtgtgtcagttagggtgtggaaagtccccaggctccccaggcaggcagaagtatgcaaagcatgcatctcaattagtcagcaaccaggtgtggaaagtccccaggctccccagcaggcagaagtatgcaaagcatgcatctcaattagtcagcaaccatagtcccgcccctaactccgcccatcccgcccctaactccgcccagttccgcccattctccgccccatggctgactaattttttttatttatgcagaggccgaggccgcctctgcctctgagctattccagaagtagtgaggaggcttttttggaggcctaggcttttgcaaaaagctccccctgttgacaattaatcatcggcatagtatatcggcatagtataatacgacaaggtgaggaactaaaccatggccaagttgaccagtgccgttccggtgctcaccgcgcgcgacgtcgccggagcggtcgagttctggaccgaccggctcgggttctcccgggacttcgtggaggacgacttcgccggtgtggtccgggacgacgtgaccctgttcatcagcgcggtccaggaccaggtggtgccggacaacaccctggcctgggtgtgggtgcgcggcctggacgagctgtacgccgagtggtcggaggtcgtgtccacgaacttccgggacgcctccgggccggccatgaccgagatcggcgagcagccgtgggggcgggagttcgccctgcgcgacccggccggcaactgcgtgcacttcgtggccgaggagcaggactgacacgtgctacgagatttaaatggtacctttaagaccaatgacttacaaggcagctgtagatcttagccactttttaaaagaaaaggggggactggaagggctaattcactcccaacgaagacaagatctgctttttgcttgtactgggtctctctggttagaccagatctgagcctgggagctctctggctaactagggaacccactgcttaagcctcaataaagcttgccttgagtgcttcaagtagtgtgtgcccgtctgttgtgtgactctggtaactagagatccctcagacccttttagtcagtgtggaaaatctctagcagtagtagttcatgtcatcttattattcagtatttataacttgcaaagaaatgaatatcagagagtgagaggaacttgtttattgcagcttataatggttacaaataaagcaatagcatcacaaatttcacaaataaagcatttttttcactgcattctagttgtggtttgtccaaactcatcaatgtatcttatcatgtctggctctagctatcccgcccctaactccgcccatcccgcccctaactccgcccagttccgcccattctccgccccatggctgactaattttttttatttatgcagaggccgaggccgcctcggcctctgagctattccagaagtagtgaggaggcttttttggaggcctagggacgtacccaattcgccctatagtgagtcgtattacgcgcgctcactggccgtcgttttacaacgtcgtgactgggaaaaccctggcgttacccaacttaatcgccttgcagcacatccccctttcgccagctggcgtaatagcgaagaggcccgcaccgatcgcccttcccaacagttgcgcagcctgaatggcgaatgggacgcgccctgtagcggcgcattaagcgcggcgggtgtggtggttacgcgcagcgtgaccgctacacttgccagcgccctagcgcccgctcctttcgctttcttcccttcctttctcgccacgttcgccggctttccccgtcaagctctaaatcgggggctccctttagggttccgatttagtgctttacggcacctcgaccccaaaaaacttgattagggtgatggttcacgtagtgggccatcgccctgatagacggtttttcgccctttgacgttggagtccacgttctttaatagtggactcttgttccaaactggaacaacactcaaccctatctcggtctattcttttgatttataagggattttgccgatttcggcctattggttaaaaaatgagctgatttaacaaaaatttaacgcgaattttaacaaaatattaacgcttacaatttaggtggcacttttcggggaaatgtgcgcggaacccctatttgtttatttttctaaatacattcaaatatgtatccgctcatgagacaataaccctgataaatgcttcaataatattgaaaaaggaagagtatgagtattcaacatttccgtgtcgcccttattcccttttttgcggcattttgccttcctgtttttgctcacccagaaacgctggtgaaagtaaaagatgctgaagatcagttgggtgcacgagtgggttacatcgaactggatctcaacagcggtaagatccttgagagttttcgccccgaagaacgttttccaatgatgagcacttttaaagttctgctatgtggcgcggtattatcccgtattgacgccgggcaagagcaactcggtcgccgcatacactattctcagaatgacttggttgagtactcaccagtcacagaaaagcatcttacggatggcatgacagtaagagaattatgcagtgctgccataaccatgagtgataacactgcggccaacttacttctgacaacgatcggaggaccgaaggagctaaccgcttttttgcacaacatgggggatcatgtaactcgccttgatcgttgggaaccggagctgaatgaagccataccaaacgacgagcgtgacaccacgatgcctgtagcaatggcaacaacgttgcgcaaactattaactggcgaactacttactctagcttcccggcaacaattaatagactggatggaggcggataaagttgcaggaccacttctgcgctcggcccttccggctggctggtttattgctgataaatctggagccggtgagcgtgggtctcgcggtatcattgcagcactggggccagatggtaagccctcccgtatcgtagttatctacacgacggggagtcaggcaactatggatgaacgaaatagacagatcgctgagataggtgcctcactgattaagcattggtaactgtcagaccaagtttactcatatatactttagattgatttaaaacttcatttttaatttaaaaggatctaggtgaagatcctttttgataatctcatgaccaaaatcccttaacgtgagttttcgttccactgagcgtcagaccccgtagaaaagatcaaaggatcttcttgagatcctttttttctgcgcgtaatctgctgcttgcaaacaaaaaaaccaccgctaccagcggtggtttgtttgccggatcaagagctaccaactctttttccgaaggtaactggcttcagcagagcgcagataccaaatactgttcttctagtgtagccgtagttaggccaccacttcaagaactctgtagcaccgcctacatacctcgctctgctaatcctgttaccagtggctgctgccagtggcgataagtcgtgtcttaccgggttggactcaagacgatagttaccggataaggcgcagcggtcgggctgaacggggggttcgtgcacacagcccagcttggagcgaacgacctacaccgaactgagatacctacagcgtgagctatgagaaagcgccacgcttcccgaagggagaaaggcggacaggtatccggtaagcggcagggtcggaacaggagagcgcacgagggagcttccagggggaaacgcctggtatctttatagtcctgtcgggtttcgccacctctgacttgagcgtcgatttttgtgatgctcgtcaggggggcggagcctatggaaaaacgccagcaacgcggcctttttacggttcctggccttttgctggccttttgctcacatgttctttcctgcgttatcccctgattctgtggataaccgtattaccgcctttgagtgagctgataccgctcgccgcagccgaacgaccgagcgcagcgagtcagtgagcgaggaagcggaagagcgcccaatacgcaaaccgcctctccccgcgcgttggccgattcattaatgcagctggcacgacaggtttcccgactggaaagcgggcagtgagcgcaacgcaattaatgtgagttagctcactcattaggcaccccaggctttacactttatgcttccggctcgtatgttgtgtggaattgtgagcggataacaatttcacacaggaaacagctatgaccatgattacgccaagcgcgcaattaaccctcactaaagggaacaaaagctggagctgcaagctt

[1260] TABLE 32 Nucleotide sequence of pLenti6/TR.aatgtagtcttatgcaatactcttgtagtcttgcaacatggtaacgatgagttagcaacatgccttacaaggagagaaaaagcaccgtgcatgccgattggtggaagtaaggtggtacgatcgtgccttattaggaaggcaacagacgggtctgacatggattggacgaaccactgaattgccgcattgcagagatattgtatttaagtgcctagctcgatacataaacgggtctctctggttagaccagatctgagcctgggagctctctggctaactagggaacccactgcttaagcctcaataaagcttgccttgagtgcttcaagtagtgtgtgcccgtctgttgtgtgactctggtaactagagatccctcagacccttttagtcagtgtggaaaatctctagcagtggcgcccgaacagggacttgaaagcgaaagggaaaccagaggagctctctcgacgcaggactcggcttgctgaagcgcgcacggcaagaggcgaggggcggcgactggtgagtacgccaaaaattttgactagcggaggctagaaggagagagatgggtgcgagagcgtcagtattaagcgggggagaattagatcgcgatgggaaaaaattcggttaaggccagggggaaagaaaaaatataaattaaaacatatagtatgggcaagcagggagctagaacgattcgcagttaatcctggcctgttagaaacatcagaaggctgtagacaaatactgggacagctacaaccatcccttcagacaggatcagaagaacttagatcattatataatacagtagcaaccctctattgtgtgcatcaaaggatagagataaaagacaccaaggaagctttagacaagatagaggaagagcaaaacaaaagtaagaccaccgcacagcaagcggccgctgatcttcagacctggaggaggagatatgagggacaattggagaagtgaattatataaatataaagtagtaaaaattgaaccattaggagtagcacccaccaaggcaaagagaagagtggtgcagagagaaaaaagagcagtgggaataggagctttgttccttgggttcttgggagcagcaggaagcactatgggcgcagcgtcaatgacgctgacggtacaggccagacaattattgtctggtatagtgcagcagcagaacaatttgctgagggctattgaggcgcaacagcatctgttgcaactcacagtctggggcatcaagcagctccaggcaagaatcctggctgtggaaagatacctaaaggatcaacagctcctggggatttggggttgctctggaaaactcatttgcaccactgctgtgccttggaatgctagttggagtaataaatctctggaacagatttggaatcacacgacctggatggagtgggacagagaaattaacaattacacaagcttaatacactccttaattgaagaatcgcaaaaccagcaagaaaagaatgaacaagaattattggaattagataaatgggcaagtttgtggaattggtttaacataacaaattggctgtggtatataaaattattcataatgatagtaggaggcttggtaggtttaagaatagtttttgctgtactttctatagtgaatagagttaggcagggatattcaccattatcgtttcagacccacctcccaaccccgaggggacccgacaggcccgaaggaatagaagaagaaggtggagagagagacagagacagatccattcgattagtgaacggatctcgacggtatcgataagcttgggagttccgcgttacataacttacggtaaatggcccgcctggctgaccgcccaacgacccccgcccattgacgtcaataatgacgtatgttcccatagtaacgccaatagggactttccattgacgtcaatgggtggagtatttacggtaaactgcccacttggcagtacatcaagtgtatcatatgccaagtacgccccctattgacgtcaatgacggtaaatggcccgcctggcattatgcccagtacatgaccttatgggactttcctacttggcagtacatctacgtattagtcatcgctattaccatggtgatgcggttttggcagtacatcaatgggcgtggatagcggtttgactcacggggatttccaagtctccaccccattgacgtcaatgggagtttgttttggcaccaaaatcaacgggactttccaaaatgtcgtaacaactccgccccattgacgcaaatgggcggtaggcgtgtacggtgggaggtctatataagcagagctcgtttagtgaaccgtcagatcgcctggagacgccatccacgctgttttgacctccatagaagacaccgactctagaggatccactagtccagtgtggtggaattctgcagatagcttggtacccggggatcctctagggcctctgagctattccagaagtagtgaagaggcttttttggaggcctaggcttttgcaaaaagctccggatcgatcctgagaacttcagggtgagtttggggacccttgattgttctttctttttcgctattgtaaaattcatgttatatggagggggcaaagttttcagggtgttgtttagaatgggaagatgtcccttgtatcaccatggaccctcatgataattttgtttctttcactttctactctgttgacaaccattgtctcctcttattttcttttcattttctgtaactttttcgttaaactttagcttgcatttgtaacgaatttttaaattcacttttgtttatttgtcagattgtaagtactttctctaatcacttttttttcaaggcaatcagggtatattatattgtacttcagcacagttttagagaacaattgttataattaaatgataaggtagaatatttctgcatataaattctggctggcgtggaaatattcttattggtagaaacaactacatcctggtcatcatcctgcctttctctttatggttacaatgatatacactgtttgagatgaggataaaatactctgagtccaaaccgggcccctctgctaaccatgttcatgccttcttctttttcctacagctcctgggcaacgtgctggttattgtgctgtctcatcattttggcaaagaattgtaatacgactcactatagggcgaattgatatgtctagattagataaaagtaaagtgattaacagcgcattagagctgcttaatgaggtcggaatcgaaggtttaacaacccgtaaactcgcccagaagctaggtgtagagcagcctacattgtattggcatgtaaaaaataagcgggctttgctcgacgccttagccattgagatgttagataggcaccatactcacttttgccctttagaaggggaaagctggcaagattttttacgtaataacgctaaaagttttagatgtgctttactaagtcatcgcgatggagcaaaagtacatttaggtacacggcctacagaaaaacagtatgaaactctcgaaaatcaattagcctttttatgccaacaaggtttttcactagagaatgcattatatgcactcagcgctgtggggcattttactttaggttgcgtattggaagatcaagagcatcaagtcgctaaagaagaaagggaaacacctactactgatagtatgccgccattattacgacaagctatcgaattatttgatcaccaaggtgcagagccagccttcttattcggccttgaattgatcatatgcggattagaaaaacaacttaaatgtgaaagtgggtccgcgtacagcggatcccgggaattctagagggcccgcggttcgaacaaaaactcatctcagaagaggatctgaatatgcataccggttagtaatgagtttggaattaattctgtggaatgtgtgtcagttagggtgtggaaagtccccaggctccccaggcaggcagaagtatgcaaagcatgcatctcaattagtcagcaaccaggtgtggaaagtccccaggctccccagcaggcagaagtatgcaaagcatgcatctcaattagtcagcaaccatagtcccgcccctaactccgcccatcccgcccctaactccgcccagttccgcccattctccgccccatggctgactaattttttttatttatgcagaggccgaggccgcctctgcctctgagctattccagaagtagtgaggaggcttttttggaggcctaggcttttgcaaaaagctcccgggagcttgtatatccattttcggatctgatcagcacgtgttgacaattaatcatcggcatagtatatcggcatagtataatacgacaaggtgaggaactaaaccatggccaagcctttgtctcaagaagaatccaccctcattgaaagagcaacggctacaatcaacagcatccccatctctgaagactacagcgtcgccagcgcagctctctctagcgacggccgcatcttcactggtgtcaatgtatatcattttactgggggaccttgtgcagaactcgtggtgctgggcactgctgctgctgcggcagctggcaacctgacttgtatcgtcgcgatcggaaatgagaacaggggcatcttgagcccctgcggacggtgccgacaggtgcttctcgatctgcatcctgggatcaaagccatagtgaaggacagtgatggacagccgacggcagttgggattcgtgaattgctgccctctggttatgtgtgggagggctaagcacaattcgagctcggtacctttaagaccaatgacttacaaggcagctgtagatcttagccactttttaaaagaaaaggggggactggaagggctaattcactcccaacgaagacaagatctgctttttgcttgtactgggtctctctggttagaccagatctgagcctgggagctctctggctaactagggaacccactgcttaagcctcaataaagcttgccttgagtgcttcaagtagtgtgtgcccgtctgttgtgtgactctggtaactagagatccctcagacccttttagtcagtgtggaaaatctctagcagtagtagttcatgtcatcttattattcagtatttataacttgcaaagaaatgaatatcagagagtgagaggaacttgtttattgcagcttataatggttacaaataaagcaatagcatcacaaatttcacaaataaagcatttttttcactgcattctagttgtggtttgtccaaactcatcaatgtatcttatcatgtctggctctagctatcccgcccctaactccgcccatcccgcccctaactccgcccagttccgcccattctccgccccatggctgactaattttttttatttatgcagaggccgaggccgcctcggcctctgagctattccagaagtagtgaggaggcttttttggaggcctagggacgtacccaattcgccctatagtgagtcgtattacgcgcgctcactggccgtcgttttacaacgtcgtgactgggaaaaccctggcgttacccaacttaatcgccttgcagcacatccccctttcgccagctggcgtaatagcgaagaggcccgcaccgatcgcccttcccaacagttgcgcagcctgaatggcgaatgggacgcgccctgtagcggcgcattaagcgcggcgggtgtggtggttacgcgcagcgtgaccgctacacttgccagcgccctagcgcccgctcctttcgctttcttcccttcctttctcgccacgttcgccggctttccccgtcaagctctaaatcgggggctccctttagggttccgatttagtgctttacggcacctcgaccccaaaaaacttgattagggtgatggttcacgtagtgggccatcgccctgatagacggtttttcgccctttgacgttggagtccacgttctttaatagtggactcttgttccaaactggaacaacactcaaccctatctcggtctattcttttgatttataagggattttgccgatttcggcctattggttaaaaaatgagctgatttaacaaaaatttaacgcgaattttaacaaaatattaacgcttacaatttaggtggcacttttcggggaaatgtgcgcggaacccctatttgtttatttttctaaatacattcaaatatgtatccgctcatgagacaataaccctgataaatgcttcaataatattgaaaaaggaagagtatgagtattcaacatttccgtgtcgcccttattcccttttttgcggcattttgccttcctgtttttgctcacccagaaacgctggtgaaagtaaaagatgctgaagatcagttgggtgcacgagtgggttacatcgaactggatctcaacagcggtaagatccttgagagttttcgccccgaagaacgttttccaatgatgagcacttttaaagttctgctatgtggcgcggtattatcccgtattgacgccgggcaagagcaactcggtcgccgcatacactattctcagaatgacttggttgagtactcaccagtcacagaaaagcatcttacggatggcatgacagtaagagaattatgcagtgctgccataaccatgagtgataacactgcggccaacttacttctgacaacgatcggaggaccgaaggagctaaccgcttttttgcacaacatgggggatcatgtaactcgccttgatcgttgggaaccggagctgaatgaagccataccaaacgacgagcgtgacaccacgatgcctgtagcaatggcaacaacgttgcgcaaactattaactggcgaactacttactctagcttcccggcaacaattaatagactggatggaggcggataaagttgcaggaccacttctgcgctcggcccttccggctggctggtttattgctgataaatctggagccggtgagcgtgggtctcgcggtatcattgcagcactggggccagatggtaagccctcccgtatcgtagttatctacacgacggggagtcaggcaactatggatgaacgaaatagacagatcgctgagataggtgcctcactgattaagcattggtaactgtcagaccaagtttactcatatatactttagattgatttaaaacttcatttttaatttaaaaggatctaggtgaagatcctttttgataatctcatgaccaaaatcccttaacgtgagttttcgttccactgagcgtcagaccccgtagaaaagatcaaaggatcttcttgagatcctttttttctgcgcgtaatctgctgcttgcaaacaaaaaaaccaccgctaccagcggtggtttgtttgccggatcaagagctaccaactctttttccgaaggtaactggcttcagcagagcgcagataccaaatactgttcttctagtgtagccgtagttaggccaccacttcaagaactctgtagcaccgcctacatacctcgctctgctaatcctgttaccagtggctgctgccagtggcgataagtcgtgtcttaccgggttggactcaagacgatagttaccggataaggcgcagcggtcgggctgaacggggggttcgtgcacacagcccagcttggagcgaacgacctacaccgaactgagatacctacagcgtgagctatgagaaagcgccacgcttcccgaagggagaaaggcggacaggtatccggtaagcggcagggtcggaacaggagagcgcacgagggagcttccagggggaaacgcctggtatctttatagtcctgtcgggtttcgccacctctgacttgagcgtcgatttttgtgatgctcgtcaggggggcggagcctatggaaaaacgccagcaacgcggcctttttacggttcctggccttttgctggccttttgctcacatgttctttcctgcgttatcccctgattctgtggataaccgtattaccgcctttgagtgagctgataccgctcgccgcagccgaacgaccgagcgcagcgagtcagtgagcgaggaagcggaagagcgcccaatacgcaaaccgcctctccccgcgcgttggccgattcattaatgcagctggcacgacaggtttcccgactggaaagcgggcagtgagcgcaacgcaattaatgtgagttagctcactcattaggcaccccaggctttacactttatgcttccggctcgtatgttgtgtggaattgtgagcggataacaatttcacacaggaaacagctatgaccatgattacgccaagcgcgcaattaaccctcactaaagggaacaaaagctggagctgcaagctt

[1261] TABLE 33 Nucleotide sequence of pLenti6/V5.aatgtagtcttatgcaatactcttgtagtcttgcaacatggtaacgatgagttagcaacatgccttacaaggagagaaaaagcaccgtgcatgccgattggtggaagtaaggtggtacgatcgtgccttattaggaaggcaacagacgggtctgacatggattggacgaaccactgaattgccgcattgcagagatattgtatttaagtgcctagctcgatacataaacgggtctctctggttagaccagatctgagcctgggagctctctggctaactagggaacccactgcttaagcctcaataaagcttgccttgagtgcttcaagtagtgtgtgcccgtctgttgtgtgactctggtaactagagatccctcagacccttttagtcagtgtggaaaatctctagcagtggcgcccgaacagggacttgaaagcgaaagggaaaccagaggagctctctcgacgcaggactcggcttgctgaagcgcgcacggcaagaggcgaggggcggcgactggtgagtacgccaaaaattttgactagcggaggctagaaggagagagatgggtgcgagagcgtcagtattaagcgggggagaattagatcgcgatgggaaaaaattcggttaaggccagggggaaagaaaaaatataaattaaaacatatagtatgggcaagcagggagctagaacgattcgcagttaatcctggcctgttagaaacatcagaaggctgtagacaaatactgggacagctacaaccatcccttcagacaggatcagaagaacttagatcattatataatacagtagcaaccctctattgtgtgcatcaaaggatagagataaaagacaccaaggaagctttagacaagatagaggaagagcaaaacaaaagtaagaccaccgcacagcaagcggccgctgatcttcagacctggaggaggagatatgagggacaattggagaagtgaattatataaatataaagtagtaaaaattgaaccattaggagtagcacccaccaaggcaaagagaagagtggtgcagagagaaaaaagagcagtgggaataggagctttgttccttgggttcttgggagcagcaggaagcactatgggcgcagcgtcaatgacgctgacggtacaggccagacaattattgtctggtatagtgcagcagcagaacaatttgctgagggctattgaggcgcaacagcatctgttgcaactcacagtctggggcatcaagcagctccaggcaagaatcctggctgtggaaagatacctaaaggatcaacagctcctggggatttggggttgctctggaaaactcatttgcaccactgctgtgccttggaatgctagttggagtaataaatctctggaacagatttggaatcacacgacctggatggagtgggacagagaaattaacaattacacaagcttaatacactccttaattgaagaatcgcaaaaccagcaagaaaagaatgaacaagaattattggaattagataaatgggcaagtttgtggaattggtttaacataacaaattggctgtggtatataaaattattcataatgatagtaggaggcttggtaggtttaagaatagtttttgctgtactttctatagtgaatagagttaggcagggatattcaccattatcgtttcagacccacctcccaaccccgaggggacccgacaggcccgaaggaatagaagaagaaggtggagagagagacagagacagatccattcgattagtgaacggatctcgacggtatcgataagcttgggagttccgcgttacataacttacggtaaatggcccgcctggctgaccgcccaacgacccccgcccattgacgtcaataatgacgtatgttcccatagtaacgccaatagggactttccattgacgtcaatgggtggagtatttacggtaaactgcccacttggcagtacatcaagtgtatcatatgccaagtacgccccctattgacgtcaatgacggtaaatggcccgcctggcattatgcccagtacatgaccttatgggactttcctacttggcagtacatctacgtattagtcatcgctattaccatggtgatgcggttttggcagtacatcaatgggcgtggatagcggtttgactcacggggatttccaagtctccaccccattgacgtcaatgggagtttgttttggcaccaaaatcaacgggactttccaaaatgtcgtaacaactccgccccattgacgcaaatgggcggtaggcgtgtacggtgggaggtctatataagcagagctcgtttagtgaaccgtcagatcgcctggagacgccatccacgctgttttgacctccatagaagacaccgactctagaggatccactagtccagtgtggtggaattctgcagatatccagcacagtggcggccgctcgagtctagagggcccgcggttcgaaggtaagcctatccctaaccctctcctcggtctcgattctacgcgtaccggttagtaatgagtttggaattaattctgtggaatgtgtgtcagttagggtgtggaaagtccccaggctccccaggcaggcagaagtatgcaaagcatgcatctcaattagtcagcaaccaggtgtggaaagtccccaggctccccagcaggcagaagtatgcaaagcatgcatctcaattagtcagcaaccatagtcccgcccctaactccgcccatcccgcccctaactccgcccagttccgcccattctccgccccatggctgactaattttttttatttatgcagaggccgaggccgcctctgcctctgagctattccagaagtagtgaggaggcttttttggaggcctaggcttttgcaaaaagctcccgggagcttgtatatccattttcggatctgatcagcacgtgttgacaattaatcatcggcatagtatatcggcatagtataatacgacaaggtgaggaactaaaccatggccaagcctttgtctcaagaagaatccaccctcattgaaagagcaacggctacaatcaacagcatccccatctctgaagactacagcgtcgccagcgcagctctctctagcgacggccgcatcttcactggtgtcaatgtatatcattttactgggggaccttgtgcagaactcgtggtgctgggcactgctgctgctgcggcagctggcaacctgacttgtatcgtcgcgatcggaaatgagaacaggggcatcttgagcccctgcggacggtgccgacaggtgcttctcgatctgcatcctgggatcaaagccatagtgaaggacagtgatggacagccgacggcagttgggattcgtgaattgctgccctctggttatgtgtgggagggctaagcacaattcgagctcggtacctttaagaccaatgacttacaaggcagctgtagatcttagccactttttaaaagaaaaggggggactggaagggctaattcactcccaacgaagacaagatctgctttttgcttgtactgggtctctctggttagaccagatctgagcctgggagctctctggctaactagggaacccactgcttaagcctcaataaagcttgccttgagtgcttcaagtagtgtgtgcccgtctgttgtgtgactctggtaactagagatccctcagacccttttagtcagtgtggaaaatctctagcagtagtagttcatgtcatcttattattcagtatttataacttgcaaagaaatgaatatcagagagtgagaggaacttgtttattgcagcttataatggttacaaataaagcaatagcatcacaaatttcacaaataaagcatttttttcactgcattctagttgtggtttgtccaaactcatcaatgtatcttatcatgtctggctctagctatcccgcccctaactccgcccatcccgcccctaactccgcccagttccgcccattctccgccccatggctgactaattttttttatttatgcagaggccgaggccgcctcggcctctgagctattccagaagtagtgaggaggcttttttggaggcctagggacgtacccaattcgccctatagtgagtcgtattacgcgcgctcactggccgtcgttttacaacgtcgtgactgggaaaaccctggcgttacccaacttaatcgccttgcagcacatccccctttcgccagctggcgtaatagcgaagaggcccgcaccgatcgcccttcccaacagttgcgcagcctgaatggcgaatgggacgcgccctgtagcggcgcattaagcgcggcgggtgtggtggttacgcgcagcgtgaccgctacacttgccagcgccctagcgcccgctcctttcgctttcttcccttcctttctcgccacgttcgccggctttccccgtcaagctctaaatcgggggctccctttagggttccgatttagtgctttacggcacctcgaccccaaaaaacttgattagggtgatggttcacgtagtgggccatcgccctgatagacggtttttcgccctttgacgttggagtccacgttctttaatagtggactcttgttccaaactggaacaacactcaaccctatctcggtctattcttttgatttataagggattttgccgatttcggcctattggttaaaaaatgagctgatttaacaaaaatttaacgcgaattttaacaaaatattaacgcttacaatttaggtggcacttttcggggaaatgtgcgcggaacccctatttgtttatttttctaaatacattcaaatatgtatccgctcatgagacaataaccctgataaatgcttcaataatattgaaaaaggaagagtatgagtattcaacatttccgtgtcgcccttattcccttttttgcggcattttgccttcctgtttttgctcacccagaaacgctggtgaaagtaaaagatgctgaagatcagttgggtgcacgagtgggttacatcgaactggatctcaacagcggtaagatccttgagagttttcgccccgaagaacgttttccaatgatgagcacttttaaagttctgctatgtggcgcggtattatcccgtattgacgccgggcaagagcaactcggtcgccgcatacactattctcagaatgacttggttgagtactcaccagtcacagaaaagcatcttacggatggcatgacagtaagagaattatgcagtgctgccataaccatgagtgataacactgcggccaacttacttctgacaacgatcggaggaccgaaggagctaaccgcttttttgcacaacatgggggatcatgtaactcgccttgatcgttgggaaccggagctgaatgaagccataccaaacgacgagcgtgacaccacgatgcctgtagcaatggcaacaacgttgcgcaaactattaactggcgaactacttactctagcttcccggcaacaattaatagactggatggaggcggataaagttgcaggaccacttctgcgctcggcccttccggctggctggtttattgctgataaatctggagccggtgagcgtgggtctcgcggtatcattgcagcactggggccagatggtaagccctcccgtatcgtagttatctacacgacggggagtcaggcaactatggatgaacgaaatagacagatcgctgagataggtgcctcactgattaagcattggtaactgtcagaccaagtttactcatatatactttagattgatttaaaacttcatttttaatttaaaaggatctaggtgaagatcctttttgataatctcatgaccaaaatcccttaacgtgagttttcgttccactgagcgtcagaccccgtagaaaagatcaaaggatcttcttgagatcctttttttctgcgcgtaatctgctgcttgcaaacaaaaaaaccaccgctaccagcggtggtttgtttgccggatcaagagctaccaactctttttccgaaggtaactggcttcagcagagcgcagataccaaatactgttcttctagtgtagccgtagttaggccaccacttcaagaactctgtagcaccgcctacatacctcgctctgctaatcctgttaccagtggctgctgccagtggcgataagtcgtgtcttaccgggttggactcaagacgatagttaccggataaggcgcagcggtcgggctgaacggggggttcgtgcacacagcccagcttggagcgaacgacctacaccgaactgagatacctacagcgtgagctatgagaaagcgccacgcttcccgaagggagaaaggcggacaggtatccggtaagcggcagggtcggaacaggagagcgcacgagggagcttccagggggaaacgcctggtatctttatagtcctgtcgggtttcgccacctctgacttgagcgtcgatttttgtgatgctcgtcaggggggcggagcctatggaaaaacgccagcaacgcggcctttttacggttcctggccttttgctggccttttgctcacatgttctttcctgcgttatcccctgattctgtggataaccgtattaccgcctttgagtgagctgataccgctcgccgcagccgaacgaccgagcgcagcgagtcagtgagcgaggaagcggaagagcgcccaatacgcaaaccgcctctccccgcgcgttggccgattcattaatgcagctggcacgacaggtttcccgactggaaagcgggcagtgagcgcaacgcaattaatgtgagttagctcactcattaggcaccccaggctttacactttatgcttccggctcgtatgttgtgtggaattgtgagcggataacaatttcacacaggaaacagctatgaccatgattacgccaagcgcgcaattaaccctcactaaagggaacaaaagctggagctgcaagctt

[1262] TABLE 34 Nucleotide sequence of pLenti3/V5-TREx.aatgtagtcttatgcaatactcttgtagtcttgcaacatggtaacgatgagttagcaacatgccttacaaggagagaaaaagcaccgtgcatgccgattggtggaagtaaggtggtacgatcgtgccttattaggaaggcaacagacgggtctgacatggattggacgaaccactgaattgccgcattgcagagatattgtatttaagtgcctagctcgatacataaacgggtctctctggttagaccagatctgagcctgggagctctctggctaactagggaacccactgcttaagcctcaataaagcttgccttgagtgcttcaagtagtgtgtgcccgtctgttgtgtgactctggtaactagagatccctcagacccttttagtcagtgtggaaaatctctagcagtggcgcccgaacagggacttgaaagcgaaagggaaaccagaggagctctctcgacgcaggactcggcttgctgaagcgcgcacggcaagaggcgaggggcggcgactggtgagtacgccaaaaattttgactagcggaggctagaaggagagagatgggtgcgagagcgtcagtattaagcgggggagaattagatcgcgatgggaaaaaattcggttaaggccagggggaaagaaaaaatataaattaaaacatatagtatgggcaagcagggagctagaacgattcgcagttaatcctggcctgttagaaacatcagaaggctgtagacaaatactgggacagctacaaccatcccttcagacaggatcagaagaacttagatcattatataatacagtagcaaccctctattgtgtgcatcaaaggatagagataaaagacaccaaggaagctttagacaagatagaggaagagcaaaacaaaagtaagaccaccgcacagcaagcggccgctgatcttcagacctggaggaggagatatgagggacaattggagaagtgaattatataaatataaagtagtaaaaattgaaccattaggagtagcacccaccaaggcaaagagaagagtggtgcagagagaaaaaagagcagtgggaataggagctttgttccttgggttcttgggagcagcaggaagcactatgggcgcagcgtcaatgacgctgacggtacaggccagacaattattgtctggtatagtgcagcagcagaacaatttgctgagggctattgaggcgcaacagcatctgttgcaactcacagtctggggcatcaagcagctccaggcaagaatcctggctgtggaaagatacctaaaggatcaacagctcctggggatttggggttgctctggaaaactcatttgcaccactgctgtgccttggaatgctagttggagtaataaatctctggaacagatttggaatcacacgacctggatggagtgggacagagaaattaacaattacacaagcttaatacactccttaattgaagaatcgcaaaaccagcaagaaaagaatgaacaagaattattggaattagataaatgggcaagtttgtggaattggtttaacataacaaattggctgtggtatataaaattattcataatgatagtaggaggcttggtaggtttaagaatagtttttgctgtactttctatagtgaatagagttaggcagggatattcaccattatcgtttcagacccacctcccaaccccgaggggacccgacaggcccgaaggaatagaagaagaaggtggagagagagacagagacagatccattcgattagtgaacggatctcgacggtatcgataagcttgggagttccgcgttacataacttacggtaaatggcccgcctggctgaccgcccaacgacccccgcccattgacgtcaataatgacgtatgttcccatagtaacgccaatagggactttccattgacgtcaatgggtggagtatttacggtaaactgcccacttggcagtacatcaagtgtatcatatgccaagtacgccccctattgacgtcaatgacggtaaatggcccgcctggcattatgcccagtacatgaccttatgggactttcctacttggcagtacatctacgtattagtcatcgctattaccatggtgatgcggttttggcagtacatcaatgggcgtggatagcggtttgactcacggggatttccaagtctccaccccattgacgtcaatgggagtttgttttggcaccaaaatcaacgggactttccaaaatgtcgtaacaactccgccccattgacgcaaatgggcggtaggcgtgtacggtgggaggtctatataagcagagctctccctatcagtgatagagatctccctatcagtgatagagatcgtcgacgagctcgtttagtgaaccgtcagatcgcctggagacgccatccacgctgttttgacctccatagaagacaccgggaccgatccagcctccggactctagaggatccctaccggtgatatcctcgagtctagagggcccgcggttcgaaggtaagcctatccctaaccctctcctcggtctcgattctacgcgtaccggttagtaatgagtttggaattaattctgtggaatgtgtgtcagttagggtgtggaaagtccccaggctccccaggcaggcagaagtatgcaaagcatgcatctcaattagtcagcaaccaggtgtggaaagtccccaggctccccagcaggcagaagtatgcaaagcatgcatctcaattagtcagcaaccatagtcccgcccctaactccgcccatcccgcccctaactccgcccagttccgcccattctccgccccatggctgactaattttttttatttatgcagaggccgaggccgcctctgcctctgagctattccagaagtagtgaggaggcttttttggaggcctaggcttttgcaaaaagctccccctgttgacaattaatcatcggcatagtatatcggcatagtataatacgacaaggtgaggaactaaaccatggcctcaattgaacaagatggattgcacgcaggttctccggccgcttgggtggagaggctattcggctatgactgggcacaacagacaatcggctgctctgatgccgccgtgttccggctgtcagcgcaggggcgcccggttctttttgtcaagaccgacctgtccggtgccctgaatgaactgcaggacgaggcagcgcggctatcgtggctggccacgacgggcgttccttgcgcagctgtgctcgacgttgtcactgaagcgggaagggactggctgctattgggcgaagtgccggggcaggatctcctgtcatctcaccttgctcctgccgagaaagtatccatcatggctgatgcaatgcggcggctgcatacgcttgatccggctacctgcccattcgaccaccaagcgaaacatcgcatcgagcgagcacgtactcggatggaagccggtcttgtcgatcaggatgatctggacgaagagcatcaggggctcgcgccagccgaactgttcgccaggctcaaggcgcgcatgcccgacggcgaggatctcgtcgtgacccatggcgatgcctgcttgccgaatatcatggtggaaaatggccgcttttctggattcatcgactgtggccggctgggtgtggcggaccgctatcaggacatagcgttggctacccgtgatattgctgaagagcttggcggcgaatgggctgaccgcttcctcgtgctttacggtatcgccgctcccgattcgcagcgcatcgccttctatcgccttcttgacgagttcttctgagcgggactctggggttcgaaatgaccgaccaagcgacgcccaacctgccatcacgagtttaaactggtacctttaagaccaatgacttacaaggcagctgtagatcttagccactttttaaaagaaaaggggggactggaagggctaattcactcccaacgaagacaagatctgctttttgcttgtactgggtctctctggttagaccagatctgagcctgggagctctctggctaactagggaacccactgcttaagcctcaataaagcttgccttgagtgcttcaagtagtgtgtgcccgtctgttgtgtgactctggtaactagagatccctcagacccttttagtcagtgtggaaaatctctagcagtagtagttcatgtcatcttattattcagtatttataacttgcaaagaaatgaatatcagagagtgagaggaacttgtttattgcagcttataatggttacaaataaagcaatagcatcacaaatttcacaaataaagcatttttttcactgcattctagttgtggtttgtccaaactcatcaatgtatcttatcatgtctggctctagctatcccgcccctaactccgcccatcccgcccctaactccgcccagttccgcccattctccgccccatggctgactaattttttttatttatgcagaggccgaggccgcctcggcctctgagctattccagaagtagtgaggaggcttttttggaggcctagggacgtacccaattcgccctatagtgagtcgtattacgcgcgctcactggccgtcgttttacaacgtcgtgactgggaaaaccctggcgttacccaacttaatcgccttgcagcacatccccctttcgccagctggcgtaatagcgaagaggcccgcaccgatcgcccttcccaacagttgcgcagcctgaatggcgaatgggacgcgccctgtagcggcgcattaagcgcggcgggtgtggtggttacgcgcagcgtgaccgctacacttgccagcgccctagcgcccgctcctttcgctttcttcccttcctttctcgccacgttcgccggctttccccgtcaagctctaaatcgggggctccctttagggttccgatttagtgctttacggcacctcgaccccaaaaaacttgattagggtgatggttcacgtagtgggccatcgccctgatagacggtttttcgccctttgacgttggagtccacgttctttaatagtggactcttgttccaaactggaacaacactcaaccctatctcggtctattcttttgatttataagggattttgccgatttcggcctattggttaaaaaatgagctgatttaacaaaaatttaacgcgaattttaacaaaatattaacgcttacaatttaggtggcacttttcggggaaatgtgcgcggaacccctatttgtttatttttctaaatacattcaaatatgtatccgctcatgagacaataaccctgataaatgcttcaataatattgaaaaaggaagagtatgagtattcaacatttccgtgtcgcccttattcccttttttgcggcattttgccttcctgtttttgctcacccagaaacgctggtgaaagtaaaagatgctgaagatcagttgggtgcacgagtgggttacatcgaactggatctcaacagcggtaagatccttgagagttttcgccccgaagaacgttttccaatgatgagcacttttaaagttctgctatgtggcgcggtattatcccgtattgacgccgggcaagagcaactcggtcgccgcatacactattctcagaatgacttggttgagtactcaccagtcacagaaaagcatcttacggatggcatgacagtaagagaattatgcagtgctgccataaccatgagtgataacactgcggccaacttacttctgacaacgatcggaggaccgaaggagctaaccgcttttttgcacaacatgggggatcatgtaactcgccttgatcgttgggaaccggagctgaatgaagccataccaaacgacgagcgtgacaccacgatgcctgtagcaatggcaacaacgttgcgcaaactattaactggcgaactacttactctagcttcccggcaacaattaatagactggatggaggcggataaagttgcaggaccacttctgcgctcggcccttccggctggctggtttattgctgataaatctggagccggtgagcgtgggtctcgcggtatcattgcagcactggggccagatggtaagccctcccgtatcgtagttatctacacgacggggagtcaggcaactatggatgaacgaaatagacagatcgctgagataggtgcctcactgattaagcattggtaactgtcagaccaagtttactcatatatactttagattgatttaaaacttcatttttaatttaaaaggatctaggtgaagatcctttttgataatctcatgaccaaaatcccttaacgtgagttttcgttccactgagcgtcagaccccgtagaaaagatcaaaggatcttcttgagatcctttttttctgcgcgtaatctgctgcttgcaaacaaaaaaaccaccgctaccagcggtggtttgtttgccggatcaagagctaccaactctttttccgaaggtaactggcttcagcagagcgcagataccaaatactgttcttctagtgtagccgtagttaggccaccacttcaagaactctgtagcaccgcctacatacctcgctctgctaatcctgttaccagtggctgctgccagtggcgataagtcgtgtcttaccgggttggactcaagacgatagttaccggataaggcgcagcggtcgggctgaacggggggttcgtgcacacagcccagcttggagcgaacgacctacaccgaactgagatacctacagcgtgagctatgagaaagcgccacgcttcccgaagggagaaaggcggacaggtatccggtaagcggcagggtcggaacaggagagcgcacgagggagcttccagggggaaacgcctggtatctttatagtcctgtcgggtttcgccacctctgacttgagcgtcgatttttgtgatgctcgtcaggggggcggagcctatggaaaaacgccagcaacgcggcctttttacggttcctggccttttgctggccttttgctcacatgttctttcctgcgttatcccctgattctgtggataaccgtattaccgcctttgagtgagctgataccgctcgccgcagccgaacgaccgagcgcagcgagtcagtgagcgaggaagcggaagagcgcccaatacgcaaaccgcctctccccgcgcgttggccgattcattaatgcagctggcacgacaggtttcccgactggaaagcgggcagtgagcgcaacgcaattaatgtgagttagctcactcattaggcaccccaggctttacactttatgcttccggctcgtatgttgtgtggaattgtgagcggataacaatttcacacaggaaacagctatgaccatgattacgccaagcgcgcaattaaccctcactaaagggaacaaaagctggagctgcaagctt

[1263] TABLE 35 Nucleotide sequence of a nucleic acid fragmentcontaining the tetracycline repressor coding sequence.agcttggtacccggggatcctctagggcctctgagctattccagaagtagtgaagaggcttttttggaggcctaggcttttgcaaaaagctccggatcgatcctgagaacttcagggtgagtttggggacccttgattgttctttctttttcgctattgtaaaattcatgttatatggagggggcaaagttttcagggtgttgtttagaatgggaagatgtcccttgtatcaccatggaccctcatgataattttgtttctttcactttctactctgttgacaaccattgtctcctcttattttcttttcattttctgtaactttttcgttaaactttagcttgcatttgtaacgaatttttaaattcacttttgtttatttgtcagattgtaagtactttctctaatcacttttttttcaaggcaatcagggtatattatattgtacttcagcacagttttagagaacaattgttataattaaatgataaggtagaatatttctgcatataaattctggctggcgtggaaatattcttattggtagaaacaactacatcctggtcatcatcctgcctttctctttatggttacaatgatatacactgtttgagatgaggataaaatactctgagtccaaaccgggcccctctgctaaccatgttcatgccttcttctttttcctacagctcctgggcaacgtgctggttattgtgctgtctcatcattttggcaaagaattgtaatacgactcactatagggcgaattgatatgtctagattagataaaagtaaagtgattaacagcgcattagagctgcttaatgaggtcggaatcgaaggtttaacaacccgtaaactcgcccagaagctaggtgtagagcagcctacattgtattggcatgtaaaaaataagcgggctttgctcgacgccttagccattgagatgttagataggcaccatactcacttttgccctttagaaggggaaagctggcaagattttttacgtaataacgctaaaagttttagatgtgctttactaagtcatcgcgatggagcaaaagtacatttaggtacacggcctacagaaaaacagtatgaaactctcgaaaatcaattagcctttttatgccaacaaggtttttcactagagaatgcattatatgcactcagcgctgtggggcattttactttaggttgcgtattggaagatcaagagcatcaagtcgctaaagaagaaagggaaacacctactactgatagtatgccgccattattacgacaagctatcgaattatttgatcaccaaggtgcagagccagccttcttattcggccttgaattgatcatatgcggattagaaaaacaacttaaatgtgaaagtgggtccgcgtacagcggatcccgggaattctagagggcccgcggttcgaacaaaaactcatctcagaagaggatctgaatatgcata

[1264] TABLE 36 Nucleotide sequence of pRRL6/V5 also referred to aspLenti6/V5. 1 aatgtagtct tatgcaatac tcttgtagtc ttgcaacatg gtaacgatgagttagcaaca 61 tgccttacaa ggagagaaaa agcaccgtgc atgccgattg gtggaagtaaggtggtacga 121 tcgtgcctta ttaggaaggc aacagacggg tctgacatgg attggacgaaccactgaatt 181 gccgcattgc agagatattg tatttaagtg cctagctcga tacaataaacgggtctctct 241 ggttagacca gatctgagcc tgggagctct ctggctaact agggaacccactgcttaagc 301 ctcaataaag cttgccttga gtgcttcaag tagtgtgtgc ccgtctgttgtgtgactctg 361 gtaactagag atccctcaga cccttttagt cagtgtggaa aatctctagcagtggcgccc 421 gaacagggac ctgaaagcga aagggaaacc agagctctct cgacgcaggactcggcttgc 481 tgaagcgcgc acggcaagag gcgaggggcg gcgactggtg agtacgccaaaaattttgac 541 tagcggaggc tagaaggaga gagatgggtg cgagagcgtc agtattaagcgggggagaat 601 tagatcgcga tgggaaaaaa ttcggttaag gccaggggga aagaaaaaatataaattaaa 661 acatatagta tgggcaagca gggagctaga acgattcgca gttaatcctggcctgttaga 721 aacatcagaa ggctgtagac aaatactggg acagctacaa ccatcccttcagacaggatc 781 agaagaactt agatcattat ataatacagt agcaaccctc tattgtgtgcatcaaaggat 841 agagataaaa gacaccaagg aagctttaga caagatagag gaagagcaaaacaaaagtaa 901 gaccaccgca cagcaagcgg ccgctgatct tcagacctgg aggaggagatatgagggaca 961 attggagaag tgaattatat aaatataaag tagtaaaaat tgaaccattaggagtagcac 1021 ccaccaaggc aaagagaaga gtggtgcaga gagaaaaaag agcagtgggaataggagctt 1081 tgttccttgg gttcttggga gcagcaggaa gcactatggg cgcagcctcaatgacgctga 1141 cggtacaggc cagacaatta ttgtctggta tagtgcagca gcagaacaatttgctgaggg 1201 ctattgaggc gcaacagcat ctgttgcaac tcacagtctg gggcatcaagcagctccagg 1261 caagaatcct ggctgtggaa agatacctaa aggatcaaca gctcctggggatttggggtt 1321 gctctggaaa actcatttgc accactgctg tgccttggaa tgctagttggagtaataaat 1381 ctctggaaca gattggaatc acacgacctg gatggagtgg gacagagaaattaacaatta 1441 cacaagctta atacactcct taattgaaga atcgcaaaac cagcaagaaaagaatgaaca 1501 agaattattg gaattagata aatgggcaag tttgtggaat tggtttaacataacaaattg 1561 gctgtggtat ataaaattat tcataatgat agtaggaggc ttggtaggtttaagaatagt 1621 ttttgctgta ctttctatag tgaatagagt taggcaggga tattcaccattatcgtttca 1681 gacccacctc ccaaccccga ggggacccga caggcccgaa ggaatagaagaagaaggtgg 1741 agagagagac agagacagat ccattcgatt agtgaacgga tctcgacggtatcgataagc 1801 ttgggagttc cgcgttacat aacttacggt aaatggcccg cctggctgaccgcccaacga 1861 cccccgccca ttgacgtcaa taatgacgta tgttcccata gtaacgccaatagggacttt 1921 ccattgacgt caatgggtgg agtatttacg gtaaactgcc cacttggcagtacatcaagt 1981 gtatcatatg ccaagtacgc cccctattga cgtcaatgac ggtaaatggcccgcctggca 2041 ttatgcccag tacatgacct tatgggactt tcctacttgg cagtacatctacgtattagt 2101 catcgctatt accatggtga tgcggttttg gcagtacatc aatgggcgtggatagcggtt 2161 tgactcacgg ggatttccaa gtctccaccc cattgacgtc aatgggagtttgttttggca 2221 ccaaaatcaa cgggactttc caaaatgtcg taacaactcc gccccattgacgcaaatggg 2281 cggtaggcgt gtacggtggg aggtctatat aagcagagct cgtttagtgaaccgtcagat 2341 cgcctggaga cgccatccac gctgttttga cctccataga agacaccgactctagaggat 2401 ccactagtcc agtgtggtgg aattctgcag atatccagca cagtggcggccgctcgagtc 2461 tagagggccc gcggttcgaa ggtaagccta tccctaaccc tctcctcggtctcgattcta 2521 cgcgtaccgg ttagtaatga gtttggcctg ctgccggctc tgcggcctcttccgcgtctt 2581 cgccttcgcc ctcagacgag tcggatctcc ctttgggccg cctccccgcctggaattaat 2641 tctgtggaat gtgtgtcagt tagggtgtgg aaagtcccca ggctccccaggcaggcagaa 2701 gtatgcaaag catgcatctc aattagtcag caaccaggtg tggaaagtccccaggctccc 2761 cagcaggcag aagtatgcaa agcatgcatc tcaattagtc agcaaccatagtcccgcccc 2821 taactccgcc catcccgccc ctaactccgc ccagttccgc ccattctccgccccatggct 2881 gactaatttt ttttatttat gcagaggccg aggccgcctc tgcctctgagctattccaga 2941 agtagtgagg aggctttttt ggaggcctag gcttttgcaa aaagctcccgggagcttgta 3001 tatccatttt cggatctgat cagcacgtgt tgacaattaa tcatcggcatagtatatcgg 3061 catagtataa tacgacaagg tgaggaacta aaccatggcc aagcctttgtctcaagaaga 3121 atccaccctc attgaaagag caacggctac aatcaacagc atccccatctctgaagacta 3181 cagcgtcgcc agcgcagctc tctctagcga cggccgcatc ttcactggtgtcaatgtata 3241 tcattttact gggggacctt gtgcagaact cgtggtgctg ggcactgctgctgctgcggc 3301 agctggcaac ctgacttgta tcgtcgcgat cggaaatgag aacaggggcatcttgagccc 3361 ctgcggacgg tgccgacagg tgcttctcga tctgcatcct gggatcaaagccatagtgaa 3421 ggacagtgat ggacagccga cggcagttgg gattcgtgaa ttgctgccctctggttatgt 3481 gtgggagggc taagcacaat tcgagctcgg tacctttaag accaatgacttacaaggcag 3541 ctgtagatct tagccacttt ttaaaagaaa aggggggact ggaagggctaattcactccc 3601 aacgaagaca agatctgctt tttgcttgta ctgggtctct ctggttagaccagatctgag 3661 cctgggagct ctctggctaa ctagggaacc cactgcttaa gcctcaataaagcttgcctt 3721 gagtycttca agtagtgtgt gcccgtctgt tgtgtgactc tggtaactagagatccctca 3781 gaccctttta gtcagtgtgg aaaatctcta gcagtagtag ttcatgtcatcttattattc 3841 agtatttata acttgcaaag aaatgaatat cagagagtga gaggaacttgtttattgcag 3901 cttataatgg ttacaaataa agcaatagca tcacaaattt cacaaataaagcattttttt 3961 cactgcattc tagttgtggt ttgtccaaac tcatcaatgt atcttatcatgtctggctct 4021 agctatcccg cccctaactc cgcccagttc cgcccattct ccgccccatggctgactaat 4081 tttttttatt tatgcagagg ccgaggccgc ctcggcctct gagctattccagaagtagtg 4141 aggaggcttt tttggaggcc taggcttttg cgtcgagacg tacccaattcgccctatagt 4201 gagtcgtatt acgcgcgctc actggccgtc gttttacaac gtcgtgactgggaaaaccct 4261 ggcgttaccc aacttaatcg ccttgcagca catccccctt tcgccagctggcgtaatagc 4321 gaagaggccc gcaccgatcg cccttcccaa cagttgcgca gcctgaatggcgaatggcgc 4381 gacgcgccct gtagcggcgc attaagcgcg gcgggtgtgg tggttacgcgcagcgtgacc 4441 gctacacttg ccagcgccct agcgcccgct cctttcgctt tcttcccttcctttctcgcc 4501 acgttcgccg gctttccccg tcaagctcta aatcgggggc tccctttagggttccgattt 4561 agtgctttac ggcacctcga ccccaaaaaa cttgattagg gtgatggttcacgtagtggg 4621 ccatcgccct gatagacggt ttttcgccct ttgacgttgg agtccacgttctttaatagt 4681 ggactcttgt tccaaactgg aacaacactc aaccctatct cggtctattcttttgattta 4741 taagggattt tgccgatttc ggcctattgg ttaaaaaatg agctgatttaacaaaaattt 4801 aacgcgaatt ttaacaaaat attaacgttt acaatttccc aggtggcacttttcggggaa 4861 atgtgcgcgg aacccctatt tgtttatttt tctaaataca ttcaaatatgtatccgctca 4921 tgagacaata accctgataa atgcttcaat aatattgaaa aaggaagagtatgagtattc 4981 aacatttccg tgtcgccctt attccctttt ttgcggcatt ttgccttcctgtttttgctc 5041 acccagaaac gctggtgaaa gtaaaagatg ctgaagatca gttgggtgcacgagtgggtt 5101 acatcgaact ggatctcaac agcggtaaga tccttgagag ttttcgccccgaagaacgtt 5161 ttccaatgat gagcactttt aaagttctgc tatgtggcgc ggtattatcccgtattgacg 5221 ccgggcaaga gcaactcggt cgccgcatac actattctca gaatgacttggttgagtact 5281 caccagtcac agaaaagcat cttacggatg gcatgacagt aagagaattatgcagtgctg 5341 ccataaccat gagtgataac actgcggcca acttacttct gacaacgatcggaggaccga 5401 aggagctaac cgcttttttg cacaacatgg gggatcatgt aactcgccttgatcgttggg 5461 aaccggagct gaatgaagcc ataccaaacg acgagcgtga caccacgatgcctgtagcaa 5521 tggcaacaac gttgcgcaaa ctattaactg gcgaactact tactctagcttcccggcaac 5581 aattaataga ctggatggag gcggataaag ttgcaggacc acttctgcgctcggcccttc 5641 cggctggctg gtttattgct gataaatctg gagccggtga gcgtgggtctcgcggtatca 5701 ttgcagcact ggggccagat ggtaagccct cccgtatcgt agttatctacacgacgggga 5761 gtcaggcaac tatggatgaa cgaaatagac agatcgctga gataggtgcctcactgatta 5821 agcattggta actgtcagac caagtttact catatatact ttagattgatttaaaacttc 5881 atttttaatt taaaaggatc taggtgaaga tcctttttga taatctcatgaccaaaatcc 5941 cttaacgtga gttttcgttc cactgagcgt cagaccccgt agaaaagatcaaaggatctt 6001 cttgagatcc tttttttctg cgcgtaatct gctgcttgca aacaaaaaaaccaccgctac 6061 cagcggtggt ttgtttgccg gatcaagagc taccaactct ttttccgaaggtaactggct 6121 tcagcagagc gcagatacca aatactgtcc ttctagtgta gccgtagttaggccaccact 6181 tcaagaactc tgtagcaccg cctacatacc tcgctctgct aatcctgttaccagtggctg 6241 ctgccagtgg cgataagtcg tgtcttaccg ggttggactc aagacgatagttaccggata 6301 aggcgcagcg gtcgggctga acggggggtt cgtgcacaca gcccagcttggagcgaacga 6361 cctacaccga actgagatac ctacagcgtg agctatgaga aagcgccacgcttcccgaag 6421 ggagaaaggc ggacaggtat ccggtaagcg gcagggtcgg aacaggagagcgcacgaggg 6481 agcttccagg gggaaacgcc tggtatcttt atagtcctgt cgggtttcgccacctctgac 6541 ttgagcgtcg atttttgtga tgctcgtcag gggggcggag cctatggaaaaacgccagca 6601 acgcggcctt tttacggttc ctggcctttt gctggccttt tgctcacatgttctttcctg 6661 cgttatcccc tgattctgtg gataaccgta ttaccgcctt tgagtgagctgataccgctc 6721 gccgcagccg aacgaccgag cgcagcgagt cagtgagcga ggaagcggaagagcgcccaa 6781 tacgcaaacc gcctctcccc gcgcgttggc cgattcatta atgcagctggcacgacaggt 6841 ttcccgactg gaaagcgggc agtgagcgca acgcaattaa tgtgagttagctcactcatt 6901 aggcacccca ggctttacac tttatgcttc cggctcgtat gttgtgtggaattgtgagcg 6961 gataacaatt tcacacagga aacagctatg accatgatta cgccaagcgcgcaattaacc 7021 ctcactaaag ggaacaaaag ctggagctgc aagctt

[1265]

1 165 1 15 DNA Unknown Core region of the wildtype att site 1 gcttttttatactaa 15 2 21 DNA Unknown Reference sequence for att site seven basepair overlap region 2 caactttttt atacaaagtt g 21 3 25 DNA ArtificialSequence attB1 site 3 agcctgcttt tttgtacaaa cttgt 25 4 233 DNAArtificial Sequence attP1 site 4 tacaggtcac taataccatc taagtagttgattcatagtg actggatatg ttgtgtttta 60 cagtattatg tagtctgttt tttatgcaaaatctaattta atatattgat atttatatca 120 ttttacgttt ctcgttcagc ttttttgtacaaagttggca ttataaaaaa gcattgctca 180 tcaatttgtt gcaacgaaca ggtcactatcagtcaaaata aaatcattat ttg 233 5 100 DNA Artificial Sequence attL1 site 5caaataatga ttttattttg actgatagtg acctgttcgt tgcaacaaat tgataagcaa 60tgctttttta taatgccaac tttgtacaaa aaagcaggct 100 6 125 DNA ArtificialSequence attR1 site 6 acaagtttgt acaaaaaagc tgaacgagaa acgtaaaatgatataaatat caatatatta 60 aattagattt tgcataaaaa acagactaca taatactgtaaaacacaaca tatccagtca 120 ctatg 125 7 27 DNA Artificial Sequence attB0site 7 agcctgcttt tttatactaa cttgagc 27 8 27 DNA Artificial SequenceattP0 site 8 gttcagcttt tttatactaa gttggca 27 9 27 DNA ArtificialSequence attL0 site 9 agcctgcttt tttatactaa gttggca 27 10 27 DNAArtificial Sequence attR0 site 10 gttcagcttt tttatactaa cttgagc 27 11 25DNA Artificial Sequence attB1 site 11 agcctgcttt tttgtacaaa cttgt 25 1227 DNA Artificial Sequence attP1 site 12 gttcagcttt tttgtacaaa gttggca27 13 27 DNA Artificial Sequence attL1 site 13 agcctgcttt tttgtacaaagttggca 27 14 25 DNA Artificial Sequence attR1 site 14 gttcagcttttttgtacaaa cttgt 25 15 25 DNA Artificial Sequence attB2 site 15acccagcttt cttgtacaaa gtggt 25 16 27 DNA Artificial Sequence attP2 site16 gttcagcttt cttgtacaaa gttggca 27 17 27 DNA Artificial Sequence attL2site 17 acccagcttt cttgtacaaa gttggca 27 18 25 DNA Artificial SequenceattR2 site 18 gttcagcttt cttgtacaaa gtggt 25 19 22 DNA ArtificialSequence attB5 site 19 caactttatt atacaaagtt gt 22 20 27 DNA ArtificialSequence attP5 site 20 gttcaacttt attatacaaa gttggca 27 21 24 DNAArtificial Sequence attL5 site 21 caactttatt atacaaagtt ggca 24 22 25DNA Artificial Sequence attR5 site 22 gttcaacttt attatacaaa gttgt 25 2322 DNA Artificial Sequence attB11 site 23 caacttttct atacaaagtt gt 22 2427 DNA Artificial Sequence attP11 site 24 gttcaacttt tctatacaaa gttggca27 25 24 DNA Artificial Sequence attL11 site 25 caacttttct atacaaagttggca 24 26 25 DNA Artificial Sequence attR11 site 26 gttcaacttttctatacaaa gttgt 25 27 22 DNA Artificial Sequence attB17 site 27caacttttgt atacaaagtt gt 22 28 27 DNA Artificial Sequence attP17 site 28gttcaacttt tgtatacaaa gttggca 27 29 24 DNA Artificial Sequence attL17site 29 caacttttgt atacaaagtt ggca 24 30 25 DNA Artificial SequenceattR17 site 30 gttcaacttt tgtatacaaa gttgt 25 31 22 DNA ArtificialSequence attB19 site 31 caactttttc gtacaaagtt gt 22 32 27 DNA ArtificialSequence attP19 site 32 gttcaacttt ttcgtacaaa gttggca 27 33 24 DNAArtificial Sequence attL19 site 33 caactttttc gtacaaagtt ggca 24 34 25DNA Artificial Sequence attR19 site 34 gttcaacttt ttcgtacaaa gttgt 25 3522 DNA Artificial Sequence attB20 site 35 caactttttg gtacaaagtt gt 22 3627 DNA Artificial Sequence attP20 site 36 gttcaacttt ttggtacaaa gttggca27 37 24 DNA Artificial Sequence attL20 site 37 caactttttg gtacaaagttggca 24 38 25 DNA Artificial Sequence attR20 site 38 gttcaactttttggtacaaa gttgt 25 39 22 DNA Artificial Sequence attB21 site 39caacttttta atacaaagtt gt 22 40 27 DNA Artificial Sequence attP21 site 40gttcaacttt ttaatacaaa gttggca 27 41 24 DNA Artificial Sequence attL21site 41 caacttttta atacaaagtt ggca 24 42 25 DNA Artificial SequenceattR21 site 42 gttcaacttt ttaatacaaa gttgt 25 43 21 DNA ArtificialSequence att system core integrase binding site 43 caactttnnn nnnnaaagttg 21 44 21 DNA Artificial Sequence attB core integrase binding site 44caactttnnn nnnnaaacaa g 21 45 20 DNA Unknown T7 promoter or priming site45 taatacgact cactataggg 20 46 21 DNA Unknown V5 reverse priming site 46accgaggaga gggttaggga t 21 47 24 DNA Unknown pAd forward priming site 47gactttgacc gtttacgtgg agac 24 48 24 DNA Unknown pAd reverse priming site48 ccttaagcca cgcccacaca tttc 24 49 14 PRT SV5 paramyxovirus 49 Gly LysPro Ile Pro Asn Pro Leu Leu Gly Leu Asp Ser Thr 1 5 10 50 6 PRTArtificial Sequence 6x His tag 50 His His His His His His 1 5 51 30 DNAUnknown pIB Neg For oligonucleotide 51 tgagtcaagg gctgccgggc tgcagcactg30 52 32 DNA Unknown pIB Neg Rev oligonucleotide 52 cggaacaagggcatgaccaa aatcccttaa cg 32 53 33 DNA Unknown gp64 For oligonucleotide53 gactcaaagg gcttgcttgt gtgttcctta ttg 33 54 34 DNA Unknown gp64s Revoligonucleotide 54 gttccgaagg gttgtgtcac gtaggccaga taac 34 55 38 DNAUnknown gp64L Rev oligonucleotide 55 gttccgaagg gaataatcga tttaagggtgtaatactc 38 56 33 DNA Unknown pe38 For oligonucleotide 56 gactcaaagggtttgcttat tggcaggctc tcc 33 57 31 DNA Unknown pe34s Rev oligonucleotide57 gttccgaagg gtatctgtcc cccactcagg c 31 58 32 DNA Unknown pe38L Revoligonucleotide 58 gttccgaagg gtaaagttga tgcggcgacg gc 32 59 27 DNAUnknown EcoRI sense primer 59 gaattccagc tgagcgccgg tcgctac 27 60 33 DNAUnknown BglII antisense primer 60 agatcttcat tcattctcac cactttgtac aag33 61 27 DNA Unknown V5/His 5 prime primer 61 agatctgggg aagcctatccctaaccc 27 62 32 DNA Unknown V5/His 3 prime primer 62 agatcttcaatggtgatggt gatgatgacc gg 32 63 28 DNA Unknown Topo D1 oligonucleotide 63aattgatccc ttcaccgaca tagtacag 28 64 12 DNA Unknown Topo D2oligonucleotide 64 ggtgaaggga tc 12 65 22 DNA Unknown Topo D6oligonucletide 65 tcgagccctt gacatagtac ag 22 66 21 DNA Unknown CMVforward primer 66 cgcaaatggg cggtaggcgt g 21 67 21 DNA Unknown V5reverse primer 67 accgaggaga gggttaggga t 21 68 22 DNA Unknown UBforward primer 68 tcagtgttag actagtaaat tg 22 69 15 DNA Unknown DNAsequence of the N-terminus of a theoretical protein 69 atgggatctg ataaa15 70 19 DNA Unknown Proposed PCR primer for theoretical protein 70caccatggga tctgataaa 19 71 27 DNA Unknown DNA sequence of the C terminusof a theoretical protein 71 aagtcggagc actcgacgac ggtgtag 27 72 17 DNAUnknown Proposed reverse PCR primer for C terminus of the theoreticalprotein 72 aaacaccgtc gtcgagt 17 73 33 DNA Unknown Sequence for theC-terminus of a theoretical protein 73 gcggttaagt cggagcactc gacgactgcatag 33 74 24 DNA Unknown Reverse primer for fusing the ORF of atheoretical protein in frame with the C terminal tag in pLenti6 V5 DTOPO 74 tgcagtcgtc gagtgctccg actt 24 75 27 DNA Unknown Reverse primerfor avoiding fusing the ORF of a theoretical protein in frame with the Cterminal tag in pLenti6 V5 D TOPO 75 ctatgcagtc gtcgagtgct ccgactt 27 7639 DNA Unknown tetO2 forward primer 76 gactcgagtc tccctatcag tgatagagatctcgaggtc 39 77 39 DNA Unknown tetO2 reverse primer 77 gacctcgagatctctatcac tgatagggag actcgagtc 39 78 24 DNA Unknown Forward PCR primer78 caccatggag aaaaaaatca ctgg 24 79 19 DNA Unknown Reverse PCR primer 79ctgctacgcc ccgccctgc 19 80 37 DNA Unknown Forward PCR primer 80caccgaattc tctagagatg tctgtgaaaa gaaacat 37 81 37 DNA Unknown ReversePCR primer 81 atataagctt actagtccgg atttcctcta cccgaga 37 82 22 DNAUnknown GFP reverse priming site 82 gggtaagctt tccgtatgta gc 22 83 36686DNA Artificial Sequence pAd CMV V5 DEST 83 catcatcaat aatataccttattttggatt gaagccaata tgataatgag ggggtggagt 60 ttgtgacgtg gcgcggggcgtgggaacggg gcgggtgacg tagtagtgtg gcggaagtgt 120 gatgttgcaa gtgtggcggaacacatgtaa gcgacggatg tggcaaaagt gacgtttttg 180 gtgtgcgccg gtgtacacaggaagtgacaa ttttcgcgcg gttttaggcg gatgttgtag 240 taaatttggg cgtaaccgagtaagatttgg ccattttcgc gggaaaactg aataagagga 300 agtgaaatct gaataattttgtgttactca tagcgcgtaa tatttgtcta gggccgcggg 360 gactttgacc gtttacgtggagactcgccc aggtgttttt ctcaggtgtt ttccgcgttc 420 cgggtcaaag ttggcgttttattattatag tcagtcgaag cttggatccg gtacctctag 480 aattctcgag cggccgctagcgacatcgga tctcccgatc ccctatggtc gactctcagt 540 acaatctgct ctgatgccgcatagttaagc cagtatctgc tccctgcttg tgtgttggag 600 gtcgctgagt agtgcgcgagcaaaatttaa gctacaacaa ggcaaggctt gaccgacaat 660 tgcatgaaga atctgcttagggttaggcgt tttgcgctgc ttcgcgatgt acgggccaga 720 tatacgcgtt gacattgattattgactagt tattaatagt aatcaattac ggggtcatta 780 gttcatagcc catatatggagttccgcgtt acataactta cggtaaatgg cccgcctggc 840 tgaccgccca acgacccccgcccattgacg tcaataatga cgtatgttcc catagtaacg 900 ccaataggga ctttccattgacgtcaatgg gtggactatt tacggtaaac tgcccacttg 960 gcagtacatc aagtgtatcatatgccaagt acgcccccta ttgacgtcaa tgacggtaaa 1020 tggcccgcct ggcattatgcccagtacatg accttatggg actttcctac ttggcagtac 1080 atctacgtat tagtcatcgctattaccatg gtgatgcggt tttggcagta catcaatggg 1140 cgtggatagc ggtttgactcacggggattt ccaagtctcc accccattga cgtcaatggg 1200 agtttgtttt ggcaccaaaatcaacgggac tttccaaaat gtcgtaacaa ctccgcccca 1260 ttgacgcaaa tgggcggtaggcgtgtacgg tgggaggtct atataagcag agctctctgg 1320 ctaactagag aacccactgcttactggctt atcgaaatta atacgactca ctatagggag 1380 acccaagctg gctagttaagctatcaacaa gtttgtacaa aaaagctgaa cgagaaacgt 1440 aaaatgatat aaatatcaatatattaaatt agattttgca taaaaaacag actacataat 1500 actgtaaaac acaacatatccagtcactat gaatcaacta cttagatggt attagtgacc 1560 tgtagtcgac cgacagccttccaaatgttc ttcgggtgat gctgccaact tagtcgaccg 1620 acagccttcc aaatgttcttctcaaacgga atcgtcgtat ccagcctact cgctattgtc 1680 ctcaatgccg tattaaatcataaaaagaaa taagaaaaag aggtgcgagc ctcttttttg 1740 tgtgacaaaa taaaaacatctacctattca tatacgctag tgtcatagtc ctgaaaatca 1800 tctgcatcaa gaacaatttcacaactctta tacttttctc ttacaagtcg ttcggcttca 1860 tctggatttt cagcctctatacttactaaa cgtgataaag tttctgtaat ttctactgta 1920 tcgacctgca gactggctgtgtataaggga gcctgacatt tatattcccc agaacatcag 1980 gttaatggcg tttttgatgtcattttcgcg gtggctgaga tcagccactt cttccccgat 2040 aacggagacc ggcacactggccatatcggt ggtcatcatg cgccagcttt catccccgat 2100 atgcaccacc gggtaaagttcacgggagac tttatctgac agcagacgtg cactggccag 2160 ggggatcacc atccgtcgcccgggcgtgtc aataatatca ctctgtacat ccacaaacag 2220 acgataacgg ctctctcttttataggtgta aaccttaaac tgcatttcac cagtccctgt 2280 tctcgtcagc aaaagagccgttcatttcaa taaaccgggc gacctcagcc atcccttcct 2340 gattttccgc tttccagcgttcggcacgca gacgacgggc ttcattctgc atggttgtgc 2400 ttaccagacc ggagatattgacatcatata tgccttgagc aactgatagc tgtcgctgtc 2460 aactgtcact gtaatacgctgcttcatagc acacctcttt ttgacatact tcgggtatac 2520 atatcagtat atattcttataccgcaaaaa tcagcgcgca aatacgcata ctgttatctg 2580 gcttttagta agccggatccacgcgattac gccccgccct gccactcatc gcagtactgt 2640 tgtaattcat taagcattctgccgacatgg aagccatcac agacggcatg atgaacctga 2700 atcgccagcg gcatcagcaccttgtcgcct tgcgtataat atttgcccat ggtgaaaacg 2760 ggggcgaaga agttgtccatattggccacg tttaaatcaa aactggtgaa actcacccag 2820 ggattggctg agacgaaaaacatattctca ataaaccctt tagggaaata ggccaggttt 2880 tcaccgtaac acgccacatcttgcgaatat atgtgtagaa actgccggaa atcgtcgtgg 2940 tattcactcc agagcgatgaaaacgtttca gtttgctcat ggaaaacggt gtaacaaggg 3000 tgaacactat cccatatcaccagctcaccg tctttcattg ccatacggaa ttccggatga 3060 gcattcatca ggcgggcaagaatgtgaata aaggccggat aaaacttgtg cttatttttc 3120 tttacggtct ttaaaaaggccgtaatatcc agctgaacgg tctggttata ggtacattga 3180 gcaactgact gaaatgcctcaaaatgttct ttacgatgcc attgggatat atcaacggtg 3240 gtatatccag tgatttttttctccatttta gcttccttag ctcctgaaaa tctcgataac 3300 tcaaaaaata cgcccggtagtgatcttatt tcattatggt gaaagttgga acctcttacg 3360 tgccgatcaa cgtctcattttcgccaaaag ttggcccagg gcttcccggt atcaacaggg 3420 acaccaggat ttatttattctgcgaagtga tcttccgtca caggtattta ttcggcgcaa 3480 agtgcgtcgg gtgatgctgccaacttagtc gactacaggt cactaatacc atctaagtag 3540 ttgattcata gtgactggatatgttgtgtt ttacagtatt atgtagtctg ttttttatgc 3600 aaaatctaat ttaatatattgatatttata tcattttacg tttctcgttc agctttcttg 3660 tacaaagtgg ttgatctagagggcccgcgg ttcgaaggta agcctatccc taaccctctc 3720 ctcggtctcg attctacgcgtaccggttag taatgagttt aaacggggga ggctaactga 3780 aacacggaag gagacaataccggaaggaac ccgcgctatg acggcaataa aaagacagaa 3840 taaaacgcac gggtgttgggtcgtttgttc ataaacgcgg ggttcggtcc cagggctggc 3900 actctgtcga taccccaccgagaccccatt ggggccaata cgcccgcgtt tcttcctttt 3960 ccccacccca ccccccaagttcgggtgaag gcccagggct cgcagccaac gtcggggcgg 4020 caggccctgc catagcagatccgattcgac agatcactga aatgtgtggg cgtggcttaa 4080 gggtgggaaa gaatatataaggtgggggtc ttatgtagtt ttgtatctgt tttgcagcag 4140 ccgccgccgc catgagcaccaactcgtttg atggaagcat tgtgagctca tatttgacaa 4200 cgcgcatgcc cccatgggccggggtgcgtc agaatgtgat gggctccagc attgatggtc 4260 gccccgtcct gcccgcaaactctactacct tgacctacga gaccgtgtct ggaacgccgt 4320 tggagactgc agcctccgccgccgcttcag ccgctgcagc caccgcccgc gggattgtga 4380 ctgactttgc tttcctgagcccgcttgcaa gcagtgcagc ttcccgttca tccgcccgcg 4440 atgacaagtt gacggctcttttggcacaat tggattcttt gacccgggaa cttaatgtcg 4500 tttctcagca gctgttggatctgcgccagc aggtttctgc cctgaaggct tcctcccctc 4560 ccaatgcggt ttaaaacataaataaaaaac cagactctgt ttggatttgg atcaagcaag 4620 tgtcttgctg tctttatttaggggttttgc gcgcgcggta ggcccgggac cagcggtctc 4680 ggtcgttgag ggtcctgtgtattttttcca ggacgtggta aaggtgactc tggatgttca 4740 gatacatggg cataagcccgtctctggggt ggaggtagca ccactgcaga gcttcatgct 4800 gcggggtggt gttgtagatgatccagtcgt agcaggagcg ctgggcgtgg tgcctaaaaa 4860 tgtctttcag tagcaagctgattgccaggg gcaggccctt ggtgtaagtg tttacaaagc 4920 ggttaagctg ggatgggtgcatacgtgggg atatgagatg catcttggac tgtattttta 4980 ggttggctat gttcccagccatatccctcc ggggattcat gttgtgcaga accaccagca 5040 cagtgtatcc ggtgcacttgggaaatttgt catgtagctt agaaggaaat gcgtggaaga 5100 acttggagac gcccttgtgacctccaagat tttccatgca ttcgtccata atgatggcaa 5160 tgggcccacg ggcggcggcctgggcgaaga tatttctggg atcactaacg tcatagttgt 5220 gttccaggat gagatcgtcataggccattt ttacaaagcg cgggcggagg gtgccagact 5280 gcggtataat ggttccatccggcccagggg cgtagttacc ctcacagatt tgcatttccc 5340 acgctttgag ttcagatggggggatcatgt ctacctgcgg ggcgatgaag aaaacggttt 5400 ccggggtagg ggagatcagctgggaagaaa gcaggttcct gagcagctgc gacttaccgc 5460 agccggtggg cccgtaaatcacacctatta ccgggtgcaa ctggtagtta agagagctgc 5520 agctgccgtc atccctgagcaggggggcca cttcgttaag catgtccctg actcgcatgt 5580 tttccctgac caaatccgccagaaggcgct cgccgcccag cgatagcagt tcttgcaagg 5640 aagcaaagtt tttcaacggtttgagaccgt ccgccgtagg catgcttttg agcgtttgac 5700 caagcagttc caggcggtcccacagctcgg tcacctgctc tacggcatct cgatccagca 5760 tatctcctcg tttcgcgggttggggcggct ttcgctgtac ggcagtagtc ggtgctcgtc 5820 cagacgggcc agggtcatgtctttccacgg gcgcagggtc ctcgtcagcg tagtctgggt 5880 cacggtgaag gggtgcgctccgggctgcgc gctggccagg gtgcgcttga ggctggtcct 5940 gctggtgctg aagcgctgccggtcttcgcc ctgcgcgtcg gccaggtagc atttgaccat 6000 ggtgtcatag tccagcccctccgcggcgtg gcccttggcg cgcagcttgc ccttggagga 6060 ggcgccgcac gaggggcagtgcagactttt gagggcgtag agcttgggcg cgagaaatac 6120 cgattccggg gagtaggcatccgcgccgca ggccccgcag acggtctcgc attccacgag 6180 ccaggtgagc tctggccgttcggggtcaaa aaccaggttt cccccatgct ttttgatgcg 6240 tttcttacct ctggtttccatgagccggtg tccacgctcg gtgacgaaaa ggctgtccgt 6300 gtccccgtat acagacttgagaggcctgtc ctcgagcggt gttccgcggt cctcctcgta 6360 tagaaactcg gaccactctgagacaaaggc tcgcgtccag gccagcacga aggaggctaa 6420 gtgggagggg tagcggtcgttgtccactag ggggtccact cgctccaggg tgtgaagaca 6480 catgtcgccc tcttcggcatcaaggaaggt gattggtttg taggtgtagg ccacgtgacc 6540 gggtgttcct gaaggggggctataaaaggg ggtgggggcg cgttcgtcct cactctcttc 6600 cgcatcgctg tctgcgagggccagctgttg gggtgagtac tccctctgaa aagcgggcat 6660 gacttctgcg ctaagattgtcagtttccaa aaacgaggag gatttgatat tcacctggcc 6720 cgcggtgatg cctttgagggtggccgcatc catctggtca gaaaagacaa tctttttgtt 6780 gtcaagcttg gtggcaaacgacccgtagag ggcgttggac agcaacttgg cgatggagcg 6840 cagggtttgg tttttgtcgcgatcggcgcg ctccttggcc gcgatgttta gctgcacgta 6900 ttcgcgcgca acgcaccgccattcgggaaa gacggtggtg cgctcgtcgg gcaccaggtg 6960 cacgcgccaa ccgcggttgtgcagggtgac aaggtcaacg ctggtggcta cctctccgcg 7020 taggcgctcg ttggtccagcagaggcggcc gcccttgcgc gagcagaatg gcggtagggg 7080 gtctagctgc gtctcgtccggggggtctgc gtccacggta aagaccccgg gcagcaggcg 7140 cgcgtcgaag tagtctatcttgcatccttg caagtctagc gcctgctgcc atgcgcgggc 7200 ggcaagcgcg cgctcgtatgggttgagtgg gggaccccat ggcatggggt gggtgagcgc 7260 ggaggcgtac atgccgcaaatgtcgtaaac gtagaggggc tctctgagta ttccaagata 7320 tgtagggtag catcttccaccgcggatgct ggcgcgcacg taatcgtata gttcgtgcga 7380 gggagcgagg aggtcgggaccgaggttgct acgggcgggc tgctctgctc ggaagactat 7440 ctgcctgaag atggcatgtgagttggatga tatggttgga cgctggaaga cgttgaagct 7500 ggcgtctgtg agacctaccgcgtcacgcac gaaggaggcg taggagtcgc gcagcttgtt 7560 gaccagctcg gcggtgacctgcacgtctag ggcgcagtag tccagggttt ccttgatgat 7620 gtcatactta tcctgtcccttttttttcca cagctcgcgg ttgaggacaa actcttcgcg 7680 gtctttccag tactcttggatcggaaaccc gtcggcctcc gaacggtaag agcctagcat 7740 gtagaactgg ttgacggcctggtaggcgca gcatcccttt tctacgggta gcgcgtatgc 7800 ctgcgcggcc ttccggagcgaggtgtgggt gagcgcaaag gtgtccctga ccatgacttt 7860 gaggtactgg tatttgaagtcagtgtcgtc gcatccgccc tgctcccaga gcaaaaagtc 7920 cgtgcgcttt ttggaacgcggatttggcag ggcgaaggtg acatcgttga agagtatctt 7980 tcccgcgcga ggcataaagttgcgtgtgat gcggaagggt cccggcacct cggaacggtt 8040 gttaattacc tgggcggcgagcacgatctc gtcaaagccg ttgatgttgt ggcccacaat 8100 gtaaagttcc aagaagcgcgggatgccctt gatggaaggc aattttttaa gttcctcgta 8160 ggtgagctct tcaggggagctgagcccgtg ctctgaaagg gcccagtctg caagatgagg 8220 gttggaagcg acgaatgagctccacaggtc acgggccatt agcatttgca ggtggtcgcg 8280 aaaggtccta aactggcgacctatggccat tttttctggg gtgatgcagt agaaggtaag 8340 cgggtcttgt tcccagcggtcccatccaag gttcgcggct aggtctcgcg cggcagtcac 8400 tagaggctca tctccgccgaacttcatgac cagcatgaag ggcacgagct gcttcccaaa 8460 ggcccccatc caagtataggtctctacatc gtaggtgaca aagagacgct cggtgcgagg 8520 atgcgagccg atcgggaagaactggatctc ccgccaccaa ttggaggagt ggctattgat 8580 gtggtgaaag tagaagtccctgcgacgggc cgaacactcg tgctggcttt tgtaaaaacg 8640 tgcgcagtac tggcagcggtgcacgggctg tacatcctgc acgaggttga cctgacgacc 8700 gcgcacaagg aagcagagtgggaatttgag cccctcgcct ggcgggtttg gctggtggtc 8760 ttctacttcg gctgcttgtccttgaccgtc tggctgctcg aggggagtta cggtggatcg 8820 gaccaccacg ccgcgcgagcccaaagtcca gatgtccgcg cgcggcggtc ggagcttgat 8880 gacaacatcg cgcagatgggagctgtccat ggtctggagc tcccgcggcg tcaggtcagg 8940 cgggagctcc tgcaggtttacctcgcatag acgggtcagg gcgcgggcta gatccaggtg 9000 atacctaatt tccaggggctggttggtggc ggcgtcgatg gcttgcaaga ggccgcatcc 9060 ccgcggcgcg actacggtaccgcgcggcgg gcggtgggcc gcgggggtgt ccttggatga 9120 tgcatctaaa agcggtgacgcgggcgagcc cccggaggta gggggggctc cggacccgcc 9180 gggagagggg gcaggggcacgtcggcgccg cgcgcgggca ggagctggtg ctgcgcgcgt 9240 aggttgctgg cgaacgcgacgacgcggcgg ttgatctcct gaatctggcg cctctgcgtg 9300 aagacgacgg gcccggtgagcttgagcctg aaagagagtt cgacagaatc aatttcggtg 9360 tcgttgacgg cggcctggcgcaaaatctcc tgcacgtctc ctgagttgtc ttgataggcg 9420 atctcggcca tgaactgctcgatctcttcc tcctggagat ctccgcgtcc ggctcgctcc 9480 acggtggcgg cgaggtcgttggaaatgcgg gccatgagct gcgagaaggc gttgaggcct 9540 ccctcgttcc agacgcggctgtagaccacg cccccttcgg catcgcgggc gcgcatgacc 9600 acctgcgcga gattgagctccacgtgccgg gcgaagacgg cgtagtttcg caggcgctga 9660 aagaggtagt tgagggtggtggcggtgtgt tctgccacga agaagtacat aacccagcgt 9720 cgcaacgtgg attcgttgatatcccccaag gcctcaaggc gctccatggc ctcgtagaag 9780 tccacggcga agttgaaaaactgggagttg cgcgccgaca cggttaactc ctcctccaga 9840 agacggatga gctcggcgacagtgtcgcgc acctcgcgct caaaggctac aggggcctct 9900 tcttcttctt caatctcctcttccataagg gcctcccctt cttcttcttc tggcggcggt 9960 gggggagggg ggacacggcggcgacgacgg cgcaccggga ggcggtcgac aaagcgctcg 10020 atcatctccc cgcggcgacggcgcatggtc tcggtgacgg cgcggccgtt ctcgcggggg 10080 cgcagttgga agacgccgcccgtcatgtcc cggttatggg ttggcggggg gctgccatgc 10140 ggcagggata cggcgctaacgatgcatctc aacaattgtt gtgtaggtac tccgccgccg 10200 agggacctga gcgagtccgcatcgaccgga tcggaaaacc tctcgagaaa ggcgtctaac 10260 cagtcacagt cgcaaggtaggctgagcacc gtggcgggcg gcagcgggcg gcggtcgggg 10320 ttgtttctgg cggaggtgctgctgatgatg taattaaagt aggcggtctt gagacggcgg 10380 atggtcgaca gaagcaccatgtccttgggt ccggcctgct gaatgcgcag gcggtcggcc 10440 atgccccagg cttcgttttgacatcggcgc aggtctttgt agtagtcttg catgagcctt 10500 tctaccggca cttcttcttctccttcctct tgtcctgcat ctcttgcatc tatcgctgcg 10560 gcggcggcgg agtttggccgtaggtggcgc cctcttcctc ccatgcgtgt gaccccgaag 10620 cccctcatcg gctgaagcagggctaggtcg gcgacaacgc gctcggctaa tatggcctgc 10680 tgcacctgcg tgagggtagactggaagtca tccatgtcca caaagcggtg gtatgcgccc 10740 gtgttgatgg tgtaagtgcagttggccata acggaccagt taacggtctg gtgacccggc 10800 tgcgagagct cggtgtacctgagacgcgag taagccctcg agtcaaatac gtagtcgttg 10860 caagtccgca ccaggtactggtatcccacc aaaaagtgcg gcggcggctg gcggtagagg 10920 ggccagcgta gggtggccggggctccgggg gcgagatctt ccaacataag gcgatgatat 10980 ccgtagatgt acctggacatccaggtgatg ccggcggcgg tggtggaggc gcgcggaaag 11040 tcgcggacgc ggttccagatgttgcgcagc ggcaaaaagt gctccatggt cgggacgctc 11100 tggccggtca ggcgcgcgcaatcgttgacg ctctagaccg tgcaaaagga gagcctgtaa 11160 gcgggcactc ttccgtggtctggtggataa attcgcaagg gtatcatggc ggacgaccgg 11220 ggttcgagcc ccgtatccggccgtccgccg tgatccatgc ggttaccgcc cgcgtgtcga 11280 acccaggtgt gcgacgtcagacaacggggg agtgctcctt ttggcttcct tccaggcgcg 11340 gcggctgctg cgctagcttttttggccact ggccgcgcgc agcgtaagcg gttaggctgg 11400 aaagcgaaag cattaagtggctcgctccct gtagccggag ggttattttc caagggttga 11460 gtcgcgggac ccccggttcgagtctcggac cggccggact gcggcgaacg ggggtttgcc 11520 tccccgtcat gcaagaccccgcttgcaaat tcctccggaa acagggacga gccccttttt 11580 tgcttttccc agatgcatccggtgctgcgg cagatgcgcc cccctcctca gcagcggcaa 11640 gagcaagagc agcggcagacatgcagggca ccctcccctc ctcctaccgc gtcaggaggg 11700 gcgacatccg cggttgacgcggcagcagat ggtgattacg aacccccgcg gcgccgggcc 11760 cggcactacc tggacttggaggagggcgag ggcctggcgc ggctaggagc gccctctcct 11820 gagcggtacc caagggtgcagctgaagcgt gatacgcgtg aggcgtacgt gccgcggcag 11880 aacctgtttc gcgaccgcgagggagaggag cccgaggaga tgcgggatcg aaagttccac 11940 gcagggcgcg agctgcggcatggcctgaat cgcgagcggt tgctgcgcga ggaggacttt 12000 gagcccgacg cgcgaaccgggattagtccc gcgcgcgcac acgtggcggc cgccgacctg 12060 gtaaccgcat acgagcagacggtgaaccag gagattaact ttcaaaaaag ctttaacaac 12120 cacgtgcgta cgcttgtggcgcgcgaggag gtggctatag gactgatgca tctgtgggac 12180 tttgtaagcg cgctggagcaaaacccaaat agcaagccgc tcatggcgca gctgttcctt 12240 atagtgcagc acagcagggacaacgaggca ttcagggatg cgctgctaaa catagtagag 12300 cccgagggcc gctggctgctcgatttgata aacatcctgc agagcatagt ggtgcaggag 12360 cgcagcttga gcctggctgacaaggtggcc gccatcaact attccatgct tagcctgggc 12420 aagttttacg cccgcaagatataccatacc ccttacgttc ccatagacaa ggaggtaaag 12480 atcgaggggt tctacatgcgcatggcgctg aaggtgctta ccttgagcga cgacctgggc 12540 gtttatcgca acgagcgcatccacaaggcc gtgagcgtga gccggcggcg cgagctcagc 12600 gaccgcgagc tgatgcacagcctgcaaagg gccctggctg gcacgggcag cggcgataga 12660 gaggccgagt cctactttgacgcgggcgct gacctgcgct gggccccaag ccgacgcgcc 12720 ctggaggcag ctggggccggacctgggctg gcggtggcac ccgcgcgcgc tggcaacgtc 12780 ggcggcgtgg aggaatatgacgaggacgat gagtacgagc cagaggacgg cgagtactaa 12840 gcggtgatgt ttctgatcagatgatgcaag acgcaacgga cccggcggtg cgggcggcgc 12900 tgcagagcca gccgtccggccttaactcca cggacgactg gcgccaggtc atggaccgca 12960 tcatgtcgct gactgcgcgcaatcctgacg cgttccggca gcagccgcag gccaaccggc 13020 tctccgcaat tctggaagcggtggtcccgg cgcgcgcaaa ccccacgcac gagaaggtgc 13080 tggcgatcgt aaacgcgctggccgaaaaca gggccatccg gcccgacgag gccggcctgg 13140 tctacgacgc gctgcttcagcgcgtggctc gttacaacag cggcaacgtg cagaccaacc 13200 tggaccggct ggtgggggatgtgcgcgagg ccgtggcgca gcgtgagcgc gcgcagcagc 13260 agggcaacct gggctccatggttgcactaa acgccttcct gagtacacag cccgccaacg 13320 tgccgcgggg acaggaggactacaccaact ttgtgagcgc actgcggcta atggtgactg 13380 agacaccgca aagtgaggtgtaccagtctg ggccagacta ttttttccag accagtagac 13440 aaggcctgca gaccgtaaacctgagccagg ctttcaaaaa cttgcagggg ctgtgggggg 13500 tgcgggctcc cacaggcgaccgcgcgaccg tgtctagctt gctgacgccc aactcgcgcc 13560 tgttgctgct gctaatagcgcccttcacgg acagtggcag cgtgtcccgg gacacatacc 13620 taggtcactt gctgacactgtaccgcgagg ccataggtca ggcgcatgtg gacgagcata 13680 ctttccagga gattacaagtgtcagccgcg cgctggggca ggaggacacg ggcagcctgg 13740 aggcaaccct aaactacctgctgaccaacc ggcggcagaa gatcccctcg ttgcacagtt 13800 taaacagcga ggaggagcgcattttgcgct acgtgcagca gagcgtgagc cttaacctga 13860 tgcgcgacgg ggtaacgcccagcgtggcgc tggacatgac cgcgcgcaac atggaaccgg 13920 gcatgtatgc ctcaaaccggccgtttatca accgcctaat ggactacttg catcgcgcgg 13980 ccgccgtgaa ccccgagtatttcaccaatg ccatcttgaa cccgcactgg ctaccgcccc 14040 ctggtttcta caccgggggattcgaggtgc ccgagggtaa cgatggattc ctctgggacg 14100 acatagacga cagcgtgttttccccgcaac cgcagaccct gctagagttg caacagcgcg 14160 agcaggcaga ggcggcgctgcgaaaggaaa gcttccgcag gccaagcagc ttgtccgatc 14220 taggcgctgc ggccccgcggtcagatgcta gtagcccatt tccaagcttg atagggtctc 14280 ttaccagcac tcgcaccacccgcccgcgcc tgctgggcga ggaggagtac ctaaacaact 14340 cgctgctgca gccgcagcgcgaaaaaaacc tgcctccggc atttcccaac aacgggatag 14400 agagcctagt ggacaagatgagtagatgga agacgtacgc gcaggagcac agggacgtgc 14460 caggcccgcg cccgcccacccgtcgtcaaa ggcacgaccg tcagcggggt ctggtgtggg 14520 aggacgatga ctcggcagacgacagcagcg tcctggattt gggagggagt ggcaacccgt 14580 ttgcgcacct tcgccccaggctggggagaa tgttttaaaa aaaaaaaagc atgatgcaaa 14640 ataaaaaact caccaaggccatggcaccga gcgttggttt tcttgtattc cccttagtat 14700 gcggcgcgcg gcgatgtatgaggaaggtcc tcctccctcc tacgagagtg tggtgagcgc 14760 ggcgccagtg gcggcggcgctgggttctcc cttcgatgct cccctggacc cgccgtttgt 14820 gcctccgcgg tacctgcggcctaccggggg gagaaacagc atccgttact ctgagttggc 14880 acccctattc gacaccacccgtgtgtacct ggtggacaac aagtcaacgg atgtggcatc 14940 cctgaactac cagaacgaccacagcaactt tctgaccacg gtcattcaaa acaatgacta 15000 cagcccgggg gaggcaagcacacagaccat caatcttgac gaccggtcgc actggggcgg 15060 cgacctgaaa accatcctgcataccaacat gccaaatgtg aacgagttca tgtttaccaa 15120 taagtttaag gcgcgggtgatggtgtcgcg cttgcctact aaggacaatc aggtggagct 15180 gaaatacgag tgggtggagttcacgctgcc cgagggcaac tactccgaga ccatgaccat 15240 agaccttatg aacaacgcgatcgtggagca ctacttgaaa gtgggcagac agaacggggt 15300 tctggaaagc gacatcggggtaaagtttga cacccgcaac ttcagactgg ggtttgaccc 15360 cgtcactggt cttgtcatgcctggggtata tacaaacgaa gccttccatc cagacatcat 15420 tttgctgcca ggatgcggggtggacttcac ccacagccgc ctgagcaact tgttgggcat 15480 ccgcaagcgg caacccttccaggagggctt taggatcacc tacgatgatc tggagggtgg 15540 taacattccc gcactgttggatgtggacgc ctaccaggcg agcttgaaag atgacaccga 15600 acagggcggg ggtggcgcaggcggcagcaa cagcagtggc agcggcgcgg aagagaactc 15660 caacgcggca gccgcggcaatgcagccggt ggaggacatg aacgatcatg ccattcgcgg 15720 cgacaccttt gccacacgggctgaggagaa gcgcgctgag gccgaagcag cggccgaagc 15780 tgccgccccc gctgcgcaacccgaggtcga gaagcctcag aagaaaccgg tgatcaaacc 15840 cctgacagag gacagcaagaaacgcagtta caacctaata agcaatgaca gcaccttcac 15900 ccagtaccgc agctggtaccttgcatacaa ctacggcgac cctcagaccg gaatccgctc 15960 atggaccctg ctttgcactcctgacgtaac ctgcggctcg gagcaggtct actggtcgtt 16020 gccagacatg atgcaagaccccgtgacctt ccgctccacg cgccagatca gcaactttcc 16080 ggtggtgggc gccgagctgttgcccgtgca ctccaagagc ttctacaacg accaggccgt 16140 ctactcccaa ctcatccgccagtttacctc tctgacccac gtgttcaatc gctttcccga 16200 gaaccagatt ttggcgcgcccgccagcccc caccatcacc accgtcagtg aaaacgttcc 16260 tgctctcaca gatcacgggacgctaccgct gcgcaacagc atcggaggag tccagcgagt 16320 gaccattact gacgccagacgccgcacctg cccctacgtt tacaaggccc tgggcatagt 16380 ctcgccgcgc gtcctatcgagccgcacttt ttgagcaagc atgtccatcc ttatatcgcc 16440 cagcaataac acaggctggggcctgcgctt cccaagcaag atgtttggcg gggccaagaa 16500 gcgctccgac caacacccagtgcgcgtgcg cgggcactac cgcgcgccct ggggcgcgca 16560 caaacgcggc cgcactgggcgcaccaccgt cgatgacgcc atcgacgcgg tggtggagga 16620 ggcgcgcaac tacacgcccacgccgccacc agtgtccaca gtggacgcgg ccattcagac 16680 cgtggtgcgc ggagcccggcgctatgctaa aatgaagaga cggcggaggc gcgtagcacg 16740 tcgccaccgc cgccgacccggcactgccgc ccaacgcgcg gcggcggccc tgcttaaccg 16800 cgcacgtcgc accggccgacgggcggccat gcgggccgct cgaaggctgg ccgcgggtat 16860 tgtcactgtg ccccccaggtccaggcgacg agcggccgcc gcagcagccg cggccattag 16920 tgctatgact cagggtcgcaggggcaacgt gtattgggtg cgcgactcgg ttagcggcct 16980 gcgcgtgccc gtgcgcacccgccccccgcg caactagatt gcaagaaaaa actacttaga 17040 ctcgtactgt tgtatgtatccagcggcggc ggcgcgcaac gaagctatgt ccaagcgcaa 17100 aatcaaagaa gagatgctccaggtcatcgc gccggagatc tatggccccc cgaagaagga 17160 agagcaggat tacaagccccgaaagctaaa gcgggtcaaa aagaaaaaga aagatgatga 17220 tgatgaactt gacgacgaggtggaactgct gcacgctacc gcgcccaggc gacgggtaca 17280 gtggaaaggt cgacgcgtaaaacgtgtttt gcgacccggc accaccgtag tctttacgcc 17340 cggtgagcgc tccacccgcacctacaagcg cgtgtatgat gaggtgtacg gcgacgagga 17400 cctgcttgag caggccaacgagcgcctcgg ggagtttgcc tacggaaagc ggcataagga 17460 catgctggcg ttgccgctggacgagggcaa cccaacacct agcctaaagc ccgtaacact 17520 gcagcaggtg ctgcccgcgcttgcaccgtc cgaagaaaag cgcggcctaa agcgcgagtc 17580 tggtgacttg gcacccaccgtgcagctgat ggtacccaag cgccagcgac tggaagatgt 17640 cttggaaaaa atgaccgtggaacctgggct ggagcccgag gtccgcgtgc ggccaatcaa 17700 gcaggtggcg ccgggactgggcgtgcagac cgtggacgtt cagataccca ctaccagtag 17760 caccagtatt gccaccgccacagagggcat ggagacacaa acgtccccgg ttgcctcagc 17820 ggtggcggat gccgcggtgcaggcggtcgc tgcggccgcg tccaagacct ctacggaggt 17880 gcaaacggac ccgtggatgtttcgcgtttc agccccccgg cgcccgcgcg gttcgaggaa 17940 gtacggcgcc gccagcgcgctactgcccga atatgcccta catccttcca ttgcgcctac 18000 ccccggctat cgtggctacacctaccgccc cagaagacga gcaactaccc gacgccgaac 18060 caccactgga acccgccgccgccgtcgccg tcgccagccc gtgctggccc cgatttccgt 18120 gcgcagggtg gctcgcgaaggaggcaggac cctggtgctg ccaacagcgc gctaccaccc 18180 cagcatcgtt taaaagccggtctttgtggt tcttgcagat atggccctca cctgccgcct 18240 ccgtttcccg gtgccgggattccgaggaag aatgcaccgt aggaggggca tggccggcca 18300 cggcctgacg ggcggcatgcgtcgtgcgca ccaccggcgg cggcgcgcgt cgcaccgtcg 18360 catgcgcggc ggtatcctgcccctccttat tccactgatc gccgcggcga ttggcgccgt 18420 gcccggaatt gcatccgtggccttgcaggc gcagagacac tgattaaaaa caagttgcat 18480 gtggaaaaat caaaataaaaagtctggact ctcacgctcg cttggtcctg taactatttt 18540 gtagaatgga agacatcaactttgcgtctc tggccccgcg acacggctcg cgcccgttca 18600 tgggaaactg gcaagatatcggcaccagca atatgagcgg tggcgccttc agctggggct 18660 cgctgtggag cggcattaaaaatttcggtt ccaccgttaa gaactatggc agcaaggcct 18720 ggaacagcag cacaggccagatgctgaggg ataagttgaa agagcaaaat ttccaacaaa 18780 aggtggtaga tggcctggcctctggcatta gcggggtggt ggacctggcc aaccaggcag 18840 tgcaaaataa gattaacagtaagcttgatc cccgccctcc cgtagaggag cctccaccgg 18900 ccgtggagac agtgtctccagaggggcgtg gcgaaaagcg tccgcgcccc gacagggaag 18960 aaactctggt gacgcaaatagacgagcctc cctcgtacga ggaggcacta aagcaaggcc 19020 tgcccaccac ccgtcccatcgcgcccatgg ctaccggagt gctgggccag cacacacccg 19080 taacgctgga cctgcctccccccgccgaca cccagcagaa acctgtgctg ccaggcccga 19140 ccgccgttgt tgtaacccgtcctagccgcg cgtccctgcg ccgcgccgcc agcggtccgc 19200 gatcgttgcg gcccgtagccagtggcaact ggcaaagcac actgaacagc atcgtgggtc 19260 tgggggtgca atccctgaagcgccgacgat gcttctgaat agctaacgtg tcgtatgtgt 19320 gtcatgtatg cgtccatgtcgccgccagag gagctgctga gccgccgcgc gcccgctttc 19380 caagatggct accccttcgatgatgccgca gtggtcttac atgcacatct cgggccagga 19440 cgcctcggag tacctgagccccgggctggt gcagtttgcc cgcgccaccg agacgtactt 19500 cagcctgaat aacaagtttagaaaccccac ggtggcgcct acgcacgacg tgaccacaga 19560 ccggtcccag cgtttgacgctgcggttcat ccctgtggac cgtgaggata ctgcgtactc 19620 gtacaaggcg cggttcaccctagctgtggg tgataaccgt gtgctggaca tggcttccac 19680 gtactttgac atccgcggcgtgctggacag gggccctact tttaagccct actctggcac 19740 tgcctacaac gccctggctcccaagggtgc cccaaatcct tgcgaatggg atgaagctgc 19800 tactgctctt gaaataaacctagaagaaga ggacgatgac aacgaagacg aagtagacga 19860 gcaagctgag cagcaaaaaactcacgtatt tgggcaggcg ccttattctg gtataaatat 19920 tacaaaggag ggtattcaaataggtgtcga aggtcaaaca cctaaatatg ccgataaaac 19980 atttcaacct gaacctcaaataggagaatc tcagtggtac gaaactgaaa ttaatcatgc 20040 agctgggaga gtccttaaaaagactacccc aatgaaacca tgttacggtt catatgcaaa 20100 acccacaaat gaaaatggagggcaaggcat tcttgtaaag caacaaaatg gaaagctaga 20160 aagtcaagtg gaaatgcaatttttctcaac tactgaggcg accgcaggca atggtgataa 20220 cttgactcct aaagtggtattgtacagtga agatgtagat atagaaaccc cagacactca 20280 tatttcttac atgcccactattaaggaagg taactcacga gaactaatgg gccaacaatc 20340 tatgcccaac aggcctaattacattgcttt tagggacaat tttattggtc taatgtatta 20400 caacagcacg ggtaatatgggtgttctggc gggccaagca tcgcagttga atgctgttgt 20460 agatttgcaa gacagaaacacagagctttc ataccagctt ttgcttgatt ccattggtga 20520 tagaaccagg tacttttctatgtggaatca ggctgttgac agctatgatc cagatgttag 20580 aattattgaa aatcatggaactgaagatga acttccaaat tactgctttc cactgggagg 20640 tgtgattaat acagagactcttaccaaggt aaaacctaaa acaggtcagg aaaatggatg 20700 ggaaaaagat gctacagaattttcagataa aaatgaaata agagttggaa ataattttgc 20760 catggaaatc aatctaaatgccaacctgtg gagaaatttc ctgtactcca acatagcgct 20820 gtatttgccc gacaagctaaagtacagtcc ttccaacgta aaaatttctg ataacccaaa 20880 cacctacgac tacatgaacaagcgagtggt ggctcccggg ttagtggact gctacattaa 20940 ccttggagca cgctggtcccttgactatat ggacaacgtc aacccattta accaccaccg 21000 caatgctggc ctgcgctaccgctcaatgtt gctgggcaat ggtcgctatg tgcccttcca 21060 catccaggtg cctcagaagttctttgccat taaaaacctc cttctcctgc cgggctcata 21120 cacctacgag tggaacttcaggaaggatgt taacatggtt ctgcagagct ccctaggaaa 21180 tgacctaagg gttgacggagccagcattaa gtttgatagc atttgccttt acgccacctt 21240 cttccccatg gcccacaacaccgcctccac gcttgaggcc atgcttagaa acgacaccaa 21300 cgaccagtcc tttaacgactatctctccgc cgccaacatg ctctacccta tacccgccaa 21360 cgctaccaac gtgcccatatccatcccctc ccgcaactgg gcggctttcc gcggctgggc 21420 cttcacgcgc cttaagactaaggaaacccc atcactgggc tcgggctacg acccttatta 21480 cacctactct ggctctataccctacctaga tggaaccttt tacctcaacc acacctttaa 21540 gaaggtggcc attacctttgactcttctgt cagctggcct ggcaatgacc gcctgcttac 21600 ccccaacgag tttgaaattaagcgctcagt tgacggggag ggttacaacg ttgcccagtg 21660 taacatgacc aaagactggttcctggtaca aatgctagct aactacaaca ttggctacca 21720 gggcttctat atcccagagagctacaagga ccgcatgtac tccttcttta gaaacttcca 21780 gcccatgagc cgtcaggtggtggatgatac taaatacaag gactaccaac aggtgggcat 21840 cctacaccaa cacaacaactctggatttgt tggctacctt gcccccacca tgcgcgaagg 21900 acaggcctac cctgctaacttcccctatcc gcttataggc aagaccgcag ttgacagcat 21960 tacccagaaa aagtttctttgcgatcgcac cctttggcgc atcccattct ccagtaactt 22020 tatgtccatg ggcgcactcacagacctggg ccaaaacctt ctctacgcca actccgccca 22080 cgcgctagac atgacttttgaggtggatcc catggacgag cccacccttc tttatgtttt 22140 gtttgaagtc tttgacgtggtccgtgtgca ccggccgcac cgcggcgtca tcgaaaccgt 22200 gtacctgcgc acgcccttctcggccggcaa cgccacaaca taaagaagca agcaacatca 22260 acaacagctg ccgccatgggctccagtgag caggaactga aagccattgt caaagatctt 22320 ggttgtgggc catattttttgggcacctat gacaagcgct ttccaggctt tgtttctcca 22380 cacaagctcg cctgcgccatagtcaatacg gccggtcgcg agactggggg cgtacactgg 22440 atggcctttg cctggaacccgcactcaaaa acatgctacc tctttgagcc ctttggcttt 22500 tctgaccagc gactcaagcaggtttaccag tttgagtacg agtcactcct gcgccgtagc 22560 gccattgctt cttcccccgaccgctgtata acgctggaaa agtccaccca aagcgtacag 22620 gggcccaact cggccgcctgtggactattc tgctgcatgt ttctccacgc ctttgccaac 22680 tggccccaaa ctcccatggatcacaacccc accatgaacc ttattaccgg ggtacccaac 22740 tccatgctca acagtccccaggtacagccc accctgcgtc gcaaccagga acagctctac 22800 agcttcctgg agcgccactcgccctacttc cgcagccaca gtgcgcagat taggagcgcc 22860 acttcttttt gtcacttgaaaaacatgtaa aaataatgta ctagagacac tttcaataaa 22920 ggcaaatgct tttatttgtacactctcggg tgattattta cccccaccct tgccgtctgc 22980 gccgtttaaa aatcaaaggggttctgccgc gcatcgctat gcgccactgg cagggacacg 23040 ttgcgatact ggtgtttagtgctccactta aactcaggca caaccatccg cggcagctcg 23100 gtgaagtttt cactccacaggctgcgcacc atcaccaacg cgtttagcag gtcgggcgcc 23160 gatatcttga agtcgcagttggggcctccg ccctgcgcgc gcgagttgcg atacacaggg 23220 ttgcagcact ggaacactatcagcgccggg tggtgcacgc tggccagcac gctcttgtcg 23280 gagatcagat ccgcgtccaggtcctccgcg ttgctcaggg cgaacggagt caactttggt 23340 agctgccttc ccaaaaagggcgcgtgccca ggctttgagt tgcactcgca ccgtagtggc 23400 atcaaaaggt gaccgtgcccggtctgggcg ttaggataca gcgcctgcat aaaagccttg 23460 atctgcttaa aagccacctgagcctttgcg ccttcagaga agaacatgcc gcaagacttg 23520 ccggaaaact gattggccggacaggccgcg tcgtgcacgc agcaccttgc gtcggtgttg 23580 gagatctgca ccacatttcggccccaccgg ttcttcacga tcttggcctt gctagactgc 23640 tccttcagcg cgcgctgcccgttttcgctc gtcacatcca tttcaatcac gtgctcctta 23700 tttatcataa tgcttccgtgtagacactta agctcgcctt cgatctcagc gcagcggtgc 23760 agccacaacg cgcagcccgtgggctcgtga tgcttgtagg tcacctctgc aaacgactgc 23820 aggtacgcct gcaggaatcgccccatcatc gtcacaaagg tcttgttgct ggtgaaggtc 23880 agctgcaacc cgcggtgctcctcgttcagc caggtcttgc atacggccgc cagagcttcc 23940 acttggtcag gcagtagtttgaagttcgcc tttagatcgt tatccacgtg gtacttgtcc 24000 atcagcgcgc gcgcagcctccatgcccttc tcccacgcag acacgatcgg cacactcagc 24060 gggttcatca ccgtaatttcactttccgct tcgctgggct cttcctcttc ctcttgcgtc 24120 cgcataccac gcgccactgggtcgtcttca ttcagccgcc gcactgtgcg cttacctcct 24180 ttgccatgct tgattagcaccggtgggttg ctgaaaccca ccatttgtag cgccacatct 24240 tctctttctt cctcgctgtccacgattacc tctggtgatg gcgggcgctc gggcttggga 24300 gaagggcgct tctttttcttcttgggcgca atggccaaat ccgccgccga ggtcgatggc 24360 cgcgggctgg gtgtgcgcggcaccagcgcg tcttgtgatg agtcttcctc gtcctcggac 24420 tcgatacgcc gcctcatccgcttttttggg ggcgcccggg gaggcggcgg cgacggggac 24480 ggggacgaca cgtcctccatggttggggga cgtcgcgccg caccgcgtcc gcgctcgggg 24540 gtggtttcgc gctgctcctcttcccgactg gccatttcct tctcctatag gcagaaaaag 24600 atcatggagt cagtcgagaagaaggacagc ctaaccgccc cctctgagtt cgccaccacc 24660 gcctccaccg atgccgccaacgcgcctacc accttccccg tcgaggcacc cccgcttgag 24720 gaggaggaag tgattatcgagcaggaccca ggttttgtaa gcgaagacga cgaggaccgc 24780 tcagtaccaa cagaggataaaaagcaagac caggacaacg cagaggcaaa cgaggaacaa 24840 gtcgggcggg gggacgaaaggcatggcgac tacctagatg tgggagacga cgtgctgttg 24900 aagcatctgc agcgccagtgcgccattatc tgcgacgcgt tgcaagagcg cagcgatgtg 24960 cccctcgcca tagcggatgtcagccttgcc tacgaacgcc acctattctc accgcgcgta 25020 ccccccaaac gccaagaaaacggcacatgc gagcccaacc cgcgcctcaa cttctacccc 25080 gtatttgccg tgccagaggtgcttgccacc tatcacatct ttttccaaaa ctgcaagata 25140 cccctatcct gccgtgccaaccgcagccga gcggacaagc agctggcctt gcggcagggc 25200 gctgtcatac ctgatatcgcctcgctcaac gaagtgccaa aaatctttga gggtcttgga 25260 cgcgacgaga agcgcgcggcaaacgctctg caacaggaaa acagcgaaaa tgaaagtcac 25320 tctggagtgt tggtggaactcgagggtgac aacgcgcgcc tagccgtact aaaacgcagc 25380 atcgaggtca cccactttgcctacccggca cttaacctac cccccaaggt catgagcaca 25440 gtcatgagtg agctgatcgtgcgccgtgcg cagcccctgg agagggatgc aaatttgcaa 25500 gaacaaacag aggagggcctacccgcagtt ggcgacgagc agctagcgcg ctggcttcaa 25560 acgcgcgagc ctgccgacttggaggagcga cgcaaactaa tgatggccgc agtgctcgtt 25620 accgtggagc ttgagtgcatgcagcggttc tttgctgacc cggagatgca gcgcaagcta 25680 gaggaaacat tgcactacacctttcgacag ggctacgtac gccaggcctg caagatctcc 25740 aacgtggagc tctgcaacctggtctcctac cttggaattt tgcacgaaaa ccgccttggg 25800 caaaacgtgc ttcattccacgctcaagggc gaggcgcgcc gcgactacgt ccgcgactgc 25860 gtttacttat ttctatgctacacctggcag acggccatgg gcgtttggca gcagtgcttg 25920 gaggagtgca acctcaaggagctgcagaaa ctgctaaagc aaaacttgaa ggacctatgg 25980 acggccttca acgagcgctccgtggccgcg cacctggcgg acatcatttt ccccgaacgc 26040 ctgcttaaaa ccctgcaacagggtctgcca gacttcacca gtcaaagcat gttgcagaac 26100 tttaggaact ttatcctagagcgctcagga atcttgcccg ccacctgctg tgcacttcct 26160 agcgactttg tgcccattaagtaccgcgaa tgccctccgc cgctttgggg ccactgctac 26220 cttctgcagc tagccaactaccttgcctac cactctgaca taatggaaga cgtgagcggt 26280 gacggtctac tggagtgtcactgtcgctgc aacctatgca ccccgcaccg ctccctggtt 26340 tgcaattcgc agctgcttaacgaaagtcaa attatcggta cctttgagct gcagggtccc 26400 tcgcctgacg aaaagtccgcggctccgggg ttgaaactca ctccggggct gtggacgtcg 26460 gcttaccttc gcaaatttgtacctgaggac taccacgccc acgagattag gttctacgaa 26520 gaccaatccc gcccgccaaatgcggagctt accgcctgcg tcattaccca gggccacatt 26580 cttggccaat tgcaagccatcaacaaagcc cgccaagagt ttctgctacg aaagggacgg 26640 ggggtttact tggacccccagtccggcgag gagctcaacc caatcccccc gccgccgcag 26700 ccctatcagc agcagccgcgggcccttgct tcccaggatg gcacccaaaa agaagctgca 26760 gctgccgccg ccacccacggacgaggagga atactgggac agtcaggcag aggaggtttt 26820 ggacgaggag gaggaggacatgatggaaga ctgggagagc ctagacgagg aagcttccga 26880 ggtcgaagag gtgtcagacgaaacaccgtc accctcggtc gcattcccct cgccggcgcc 26940 ccagaaatcg gcaaccggttccagcatggc tacaacctcc gctcctcagg cgccgccggc 27000 actgcccgtt cgccgacccaaccgtagatg ggacaccact ggaaccaggg ccggtaagtc 27060 caagcagccg ccgccgttagcccaagagca acaacagcgc caaggctacc gctcatggcg 27120 cgggcacaag aacgccatagttgcttgctt gcaagactgt gggggcaaca tctccttcgc 27180 ccgccgcttt cttctctaccatcacggcgt ggccttcccc cgtaacatcc tgcattacta 27240 ccgtcatctc tacagcccatactgcaccgg cggcagcggc agcggcagca acagcagcgg 27300 ccacacagaa gcaaaggcgaccggatagca agactctgac aaagcccaag aaatccacag 27360 cggcggcagc agcaggaggaggagcgctgc gtctggcgcc caacgaaccc gtatcgaccc 27420 gcgagcttag aaacaggatttttcccactc tgtatgctat atttcaacag agcaggggcc 27480 aagaacaaga gctgaaaataaaaaacaggt ctctgcgatc cctcacccgc agctgcctgt 27540 atcacaaaag cgaagatcagcttcggcgca cgctggaaga cgcggaggct ctcttcagta 27600 aatactgcgc gctgactcttaaggactagt ttcgcgccct ttctcaaatt taagcgcgaa 27660 aactacgtca tctccagcggccacacccgg cgccagcacc tgtcgtcagc gccattatga 27720 gcaaggaaat tcccacgccctacatgtgga gttaccagcc acaaatggga cttgcggctg 27780 gagctgccca agactactcaacccgaataa actacatgag cgcgggaccc cacatgatat 27840 cccgggtcaa cggaatccgcgcccaccgaa accgaattct cttggaacag gcggctatta 27900 ccaccacacc tcgtaataaccttaatcccc gtagttggcc cgctgccctg gtgtaccagg 27960 aaagtcccgc tcccaccactgtggtacttc ccagagacgc ccaggccgaa gttcagatga 28020 ctaactcagg ggcgcagcttgcgggcggct ttcgtcacag ggtgcggtcg cccgggcagg 28080 gtataactca cctgacaatcagagggcgag gtattcagct caacgacgag tcggtgagct 28140 cctcgcttgg tctccgtccggacgggacat ttcagatcgg cggcgccggc cgtccttcat 28200 tcacgcctcg tcaggcaatcctaactctgc agacctcgtc ctctgagccg cgctctggag 28260 gcattggaac tctgcaatttattgaggagt ttgtgccatc ggtctacttt aaccccttct 28320 cgggacctcc cggccactatccggatcaat ttattcctaa ctttgacgcg gtaaaggact 28380 cggcggacgg ctacgactgaatgttaagtg gagaggcaga gcaactgcgc ctgaaacacc 28440 tggtccactg tcgccgccacaagtgctttg cccgcgactc cggtgagttt tgctactttg 28500 aattgcccga ggatcatatcgagggcccgg cgcacggcgt ccggcttacc gcccagggag 28560 agcttgcccg tagcctgattcgggagttta cccagcgccc cctgctagtt gagcgggaca 28620 ggggaccctg tgttctcactgtgatttgca actgtcctaa ccttggatta catcaagatc 28680 tttgttgcca tctctgtgctgagtataata aatacagaaa ttaaaatata ctggggctcc 28740 tatcgccatc ctgtaaacgccaccgtcttc acccgcccaa gcaaaccaag gcgaacctta 28800 cctggtactt ttaacatctctccctctgtg atttacaaca gtttcaaccc agacggagtg 28860 agtctacgag agaacctctccgagctcagc tactccatca gaaaaaacac caccctcctt 28920 acctgccggg aacgtacgagtgcgtcaccg gccgctgcac cacacctacc gcctgaccgt 28980 aaaccagact ttttccggacagacctcaat aactctgttt accagaacag gaggtgagct 29040 tagaaaaccc ttagggtattaggccaaagg cgcagctact gtggggttta tgaacaattc 29100 aagcaactct acgggctattctaattcagg tttctctaga aatggacgga attattacag 29160 agcagcgcct gctagaaagacgcagggcag cggccgagca acagcgcatg aatcaagagc 29220 tccaagacat ggttaacttgcaccagtgca aaaggggtat cttttgtctg gtaaagcagg 29280 ccaaagtcac ctacgacagtaataccaccg gacaccgcct tagctacaag ttgccaacca 29340 agcgtcagaa attggtggtcatggtgggag aaaagcccat taccataact cagcactcgg 29400 tagaaaccga aggctgcattcactcacctt gtcaaggacc tgaggatctc tgcaccctta 29460 ttaagaccct gtgcggtctcaaagatctta ttccctttaa ctaataaaaa aaaataataa 29520 agcatcactt acttaaaatcagttagcaaa tttctgtcca gtttattcag cagcacctcc 29580 ttgccctcct cccagctctggtattgcagc ttcctcctgg ctgcaaactt tctccacaat 29640 ctaaatggaa tgtcagtttcctcctgttcc tgtccatccg cacccactat cttcatgttg 29700 ttgcagatga agcgcgcaagaccgtctgaa gataccttca accccgtgta tccatatgac 29760 acggaaaccg gtcctccaactgtgcctttt cttactcctc cctttgtatc ccccaatggg 29820 tttcaagaga gtccccctggggtactctct ttgcgcctat ccgaacctct agttacctcc 29880 aatggcatgc ttgcgctcaaaatgggcaac ggcctctctc tggacgaggc cggcaacctt 29940 acctcccaaa atgtaaccactgtgagccca cctctcaaaa aaaccaagtc aaacataaac 30000 ctggaaatat ctgcacccctcacagttacc tcagaagccc taactgtggc tgccgccgca 30060 cctctaatgg tcgcgggcaacacactcacc atgcaatcac aggccccgct aaccgtgcac 30120 gactccaaac ttagcattgccacccaagga cccctcacag tgtcagaagg aaagctagcc 30180 ctgcaaacat caggccccctcaccaccacc gatagcagta cccttactat cactgcctca 30240 ccccctctaa ctactgccactggtagcttg ggcattgact tgaaagagcc catttataca 30300 caaaatggaa aactaggactaaagtacggg gctcctttgc atgtaacaga cgacctaaac 30360 actttgaccg tagcaactggtccaggtgtg actattaata atacttcctt gcaaactaaa 30420 gttactggag ccttgggttttgattcacaa ggcaatatgc aacttaatgt agcaggagga 30480 ctaaggattg attctcaaaacagacgcctt atacttgatg ttagttatcc gtttgatgct 30540 caaaaccaac taaatctaagactaggacag ggccctcttt ttataaactc agcccacaac 30600 ttggatatta actacaacaaaggcctttac ttgtttacag cttcaaacaa ttccaaaaag 30660 cttgaggtta acctaagcactgccaagggg ttgatgtttg acgctacagc catagccatt 30720 aatgcaggag atgggcttgaatttggttca cctaatgcac caaacacaaa tcccctcaaa 30780 acaaaaattg gccatggcctagaatttgat tcaaacaagg ctatggttcc taaactagga 30840 actggcctta gttttgacagcacaggtgcc attacagtag gaaacaaaaa taatgataag 30900 ctaactttgt ggaccacaccagctccatct cctaactgta gactaaatgc agagaaagat 30960 gctaaactca ctttggtcttaacaaaatgt ggcagtcaaa tacttgctac agtttcagtt 31020 ttggctgtta aaggcagtttggctccaata tctggaacag ttcaaagtgc tcatcttatt 31080 ataagatttg acgaaaatggagtgctacta aacaattcct tcctggaccc agaatattgg 31140 aactttagaa atggagatcttactgaaggc acagcctata caaacgctgt tggatttatg 31200 cctaacctat cagcttatccaaaatctcac ggtaaaactg ccaaaagtaa cattgtcagt 31260 caagtttact taaacggagacaaaactaaa cctgtaacac taaccattac actaaacggt 31320 acacaggaaa caggagacacaactccaagt gcatactcta tgtcattttc atgggactgg 31380 tctggccaca actacattaatgaaatattt gccacatcct cttacacttt ttcatacatt 31440 gcccaagaat aaagaatcgtttgtgttatg tttcaacgtg tttatttttc aattgcagaa 31500 aatttcgaat catttttcattcagtagtat agccccacca ccacatagct tatacagatc 31560 accgtacctt aatcaaactcacagaaccct agtattcaac ctgccacctc cctcccaaca 31620 cacagagtac acagtcctttctccccggct ggccttaaaa agcatcatat catgggtaac 31680 agacatattc ttaggtgttatattccacac ggtttcctgt cgagccaaac gctcatcagt 31740 gatattaata aactccccgggcagctcact taagttcatg tcgctgtcca gctgctgagc 31800 cacaggctgc tgtccaacttgcggttgctt aacgggcggc gaaggagaag tccacgccta 31860 catgggggta gagtcataatcgtgcatcag gatagggcgg tggtgctgca gcagcgcgcg 31920 aataaactgc tgccgccgccgctccgtcct gcaggaatac aacatggcag tggtctcctc 31980 agcgatgatt cgcaccgcccgcagcataag gcgccttgtc ctccgggcac agcagcgcac 32040 cctgatctca cttaaatcagcacagtaact gcagcacagc accacaatat tgttcaaaat 32100 cccacagtgc aaggcgctgtatccaaagct catggcgggg accacagaac ccacgtggcc 32160 atcataccac aagcgcaggtagattaagtg gcgacccctc ataaacacgc tggacataaa 32220 cattacctct tttggcatgttgtaattcac cacctcccgg taccatataa acctctgatt 32280 aaacatggcg ccatccaccaccatcctaaa ccagctggcc aaaacctgcc cgccggctat 32340 acactgcagg gaaccgggactggaacaatg acagtggaga gcccaggact cgtaaccatg 32400 gatcatcatg ctcgtcatgatatcaatgtt ggcacaacac aggcacacgt gcatacactt 32460 cctcaggatt acaagctcctcccgcgttag aaccatatcc cagggaacaa cccattcctg 32520 aatcagcgta aatcccacactgcagggaag acctcgcacg taactcacgt tgtgcattgt 32580 caaagtgtta cattcgggcagcagcggatg atcctccagt atggtagcgc gggtttctgt 32640 ctcaaaagga ggtagacgatccctactgta cggagtgcgc cgagacaacc gagatcgtgt 32700 tggtcgtagt gtcatgccaaatggaacgcc ggacgtagtc atatttcctg aagcaaaacc 32760 aggtgcgggc gtgacaaacagatctgcgtc tccggtctcg ccgcttagat cgctctgtgt 32820 agtagttgta gtatatccactctctcaaag catccaggcg ccccctggct tcgggttcta 32880 tgtaaactcc ttcatgcgccgctgccctga taacatccac caccgcagaa taagccacac 32940 ccagccaacc tacacattcgttctgcgagt cacacacggg aggagcggga agagctggaa 33000 gaaccatgtt tttttttttattccaaaaga ttatccaaaa cctcaaaatg aagatctatt 33060 aagtgaacgc gctcccctccggtggcgtgg tcaaactcta cagccaaaga acagataatg 33120 gcatttgtaa gatgttgcacaatggcttcc aaaaggcaaa cggccctcac gtccaagtgg 33180 acgtaaaggc taaacccttcagggtgaatc tcctctataa acattccagc accttcaacc 33240 atgcccaaat aattctcatctcgccacctt ctcaatatat ctctaagcaa atcccgaata 33300 ttaagtccgg ccattgtaaaaatctgctcc agagcgccct ccaccttcag cctcaagcag 33360 cgaatcatga ttgcaaaaattcaggttcct cacagacctg tataagattc aaaagcggaa 33420 cattaacaaa aataccgcgatcccgtaggt cccttcgcag ggccagctga acataatcgt 33480 gcaggtctgc acggaccagcgcggccactt ccccgccagg aaccttgaca aaagaaccca 33540 cactgattat gacacgcatactcggagcta tgctaaccag cgtagccccg atgtaagctt 33600 tgttgcatgg gcggcgatataaaatgcaag gtgctgctca aaaaatcagg caaagcctcg 33660 cgcaaaaaag aaagcacatcgtagtcatgc tcatgcagat aaaggcaggt aagctccgga 33720 accaccacag aaaaagacaccatttttctc tcaaacatgt ctgcgggttt ctgcataaac 33780 acaaaataaa ataacaaaaaaacatttaaa cattagaagc ctgtcttaca acaggaaaaa 33840 caacccttat aagcataagacggactacgg ccatgccggc gtgaccgtaa aaaaactggt 33900 caccgtgatt aaaaagcaccaccgacagct cctcggtcat gtccggagtc ataatgtaag 33960 actcggtaaa cacatcaggttgattcacat cggtcagtgc taaaaagcga ccgaaatagc 34020 ccgggggaat acatacccgcaggcgtagag acaacattac agcccccata ggaggtataa 34080 caaaattaat aggagagaaaaacacataaa cacctgaaaa accctcctgc ctaggcaaaa 34140 tagcaccctc ccgctccagaacaacataca gcgcttccac agcggcagcc ataacagtca 34200 gccttaccag taaaaaagaaaacctattaa aaaaacacca ctcgacacgg caccagctca 34260 atcagtcaca gtgtaaaaaagggccaagtg cagagcgagt atatatagga ctaaaaaatg 34320 acgtaacggt taaagtccacaaaaaacacc cagaaaaccg cacgcgaacc tacgcccaga 34380 aacgaaagcc aaaaaacccacaacttcctc aaatcgtcac ttccgttttc ccacgttacg 34440 tcacttccca ttttaagaaaactacaattc ccaacacata caagttactc cgccctaaaa 34500 cctacgtcac ccgccccgttcccacgcccc gcgccacgtc acaaactcca ccccctcatt 34560 atcatattgg cttcaatccaaaataaggta tattattgat gatgttaatt aatttaaatc 34620 cgcatgcgat atcgagctctcccgggaatt cggatctgcg acgcgaggct ggatggcctt 34680 ccccattatg attcttctcgcttccggcgg catcgggatg cccgcgttgc aggccatgct 34740 gtccaggcag gtagatgacgaccatcaggg acagcttcac ggccagcaaa aggccaggaa 34800 ccgtaaaaag gccgcgttgctggcgttttt ccataggctc cgcccccctg acgagcatca 34860 caaaaatcga cgctcaagtcagaggtggcg aaacccgaca ggactataaa gataccaggc 34920 gtttccccct ggaagctccctcgtgcgctc tcctgttccg accctgccgc ttaccggata 34980 cctgtccgcc tttctcccttcgggaagcgt ggcgctttct caatgctcac gctgtaggta 35040 tctcagttcg gtgtaggtcgttcgctccaa gctgggctgt gtgcacgaac cccccgttca 35100 gcccgaccgc tgcgccttatccggtaacta tcgtcttgag tccaacccgg taagacacga 35160 cttatcgcca ctggcagcagccactggtaa caggattagc agagcgaggt atgtaggcgg 35220 tgctacagag ttcttgaagtggtggcctaa ctacggctac actagaagga cagtatttgg 35280 tatctgcgct ctgctgaagccagttacctt cggaaaaaga gttggtagct cttgatccgg 35340 caaacaaacc accgctggtagcggtggttt ttttgtttgc aagcagcaga ttacgcgcag 35400 aaaaaaagga tctcaagaagatcctttgat cttttctacg gggtctgacg ctcagtggaa 35460 cgaaaactca cgttaagggattttggtcat gagattatca aaaaggatct tcacctagat 35520 ccttttaaat caatctaaagtatatatgag taaacttggt ctgacagtta ccaatgctta 35580 atcagtgagg cacctatctcagcgatctgt ctatttcgtt catccatagt tgcctgactc 35640 cccgtcgtgt agataactacgatacgggag ggcttaccat ctggccccag tgctgcaatg 35700 ataccgcgag acccacgctcaccggctcca gatttatcag caataaacca gccagccgga 35760 agggccgagc gcagaagtggtcctgcaact ttatccgcct ccatccagtc tattaattgt 35820 tgccgggaag ctagagtaagtagttcgcca gttaatagtt tgcgcaacgt tgttgccatt 35880 gntgcaggca tcgtggtgtcacgctcgtcg tttggtatgg cttcattcag ctccggttcc 35940 caacgatcaa ggcgagttacatgatccccc atgttgtgca aaaaagcggt tagctccttc 36000 ggtcctccga tcgttgtcagaagtaagttg gccgcagtgt tatcactcat ggttatggca 36060 gcactgcata attctcttactgtcatgcca tccgtaagat gcttttctgt gactggtgag 36120 tactcaacca agtcattctgagaatagtgt atgcggcgac cgagttgctc ttgcccggcg 36180 tcaacacggg ataataccgcgccacatagc agaactttaa aagtgctcat cattggaaaa 36240 cgttcttcgg ggcgaaaactctcaaggatc ttaccgctgt tgagatccag ttcgatgtaa 36300 cccactcgtg cacccaactgatcttcagca tcttttactt tcaccagcgt ttctgggtga 36360 gcaaaaacag gaaggcaaaatgccgcaaaa aagggaataa gggcgacacg gaaatgttga 36420 atactcatac tcttcctttttcaatattat tgaagcattt atcagggtta ttgtctcatg 36480 agcggataca tatttgaatgtatttagaaa aataaacaaa taggggttcc gcgcacattt 36540 ccccgaaaag tgccacctgacgtctaagaa accattatta tcatgacatt aacctataaa 36600 aataggcgta tcacgaggccctttcgtctt caaggatccg aattcccggg agagctcgat 36660 atcgcatgcg gatttaaattaattaa 36686 84 34226 DNA Artificial Sequence pAd GW TO tRNA 84catcatcaat aatatacctt attttggatt gaagccaata tgataatgag ggggtggagt 60ttgtgacgtg gcgcggggcg tgggaacggg gcgggtgacg tagtagtgtg gcggaagtgt 120gatgttgcaa gtgtggcgga acacatgtaa gcgacggatg tggcaaaagt gacgtttttg 180gtgtgcgccg gtgtacacag gaagtgacaa ttttcgcgcg gttttaggcg gatgttgtag 240taaatttggg cgtaaccgag taagatttgg ccattttcgc gggaaaactg aataagagga 300agtgaaatct gaataatttt gtgttactca tagcgcgtaa tatttgtcta gggccgcggg 360gactttgacc gtttacgtgg agactcgccc aggtgttttt ctcaggtgtt ttccgcgttc 420cgggtcaaag ttggcgtttt attattatag tcagtcgaag cttggatccg gtacctctag 480aattctcgag cggccgctag cgacatcgat cacaagtttg tacaaaaaag caggctttaa 540aggaaccaat tcagtcgact ctagaggatc gaaaccatcc tctgctatat ggccgcatat 600attttacttg aagactagga ccctacagaa aaggggtttt aaagtaggcg tgctaaacgt 660cagcggacct gacccgtgta agaatccaca aggtatcctg gtggaaatgc gcatttgtag 720gcttcaatat ctgtaatcct actaattagg tgtggagagc tttcagccag tttcgtaggt 780ttggagacca tttaggggtt ggcgtgtggc cccctcgtaa agtctttcgt acttcctaca 840tcagacaagt cttgcaattt gcaatatctc ttttagccaa tatctaaatc tttaaaattt 900tgattttgtt ttttacccag gatgagagac attccagagt tgttaccttg tcaaaataaa 960caaatttaaa gatgtctgtg aaaagaaaca tatattcctc atgggaatat atccaggttg 1020ttgaaggagg tacgacctcg agatctctat cactgatagg gagactcgag tgtagtcgtg 1080gccgagtggt taaggcgatg gactctaaat ccattggggt ctccccgcgc aggttcgaat 1140cctgccgact acggcgtgct ttttttactc tcgggtagag gaaatccggt gcactacctg 1200tgcaatcaca cagaataaca tggagtagta ctttttattt tcctgttatt atctttctcc 1260ataaaagtgg aaccagataa ttttagttct tttgtgtaac aagactagag attttttgaa 1320gtgttacatt ggaaagcact tgaaaacaca agtaatttct gacactgcta taaaaatgat 1380ggaaaaacgc tcaagttgtt ttgcctttca gtcttcttga aatgctgtct ccctatctga 1440aatccagctc acgtctgact tccaaaaccg tgcttgcctt taacttatgg aataaatatc 1500tcaaacagat ccccgggcga gctcgaattc gcggccgcac tcgagatatc tagacccagc 1560tttcttgtac aaagtggtga tcgattcgac agatcactga aatgtgtggg cgtggcttaa 1620gggtgggaaa gaatatataa ggtgggggtc ttatgtagtt ttgtatctgt tttgcagcag 1680ccgccgccgc catgagcacc aactcgtttg atggaagcat tgtgagctca tatttgacaa 1740cgcgcatgcc cccatgggcc ggggtgcgtc agaatgtgat gggctccagc attgatggtc 1800gccccgtcct gcccgcaaac tctactacct tgacctacga gaccgtgtct ggaacgccgt 1860tggagactgc agcctccgcc gccgcttcag ccgctgcagc caccgcccgc gggattgtga 1920ctgactttgc tttcctgagc ccgcttgcaa gcagtgcagc ttcccgttca tccgcccgcg 1980atgacaagtt gacggctctt ttggcacaat tggattcttt gacccgggaa cttaatgtcg 2040tttctcagca gctgttggat ctgcgccagc aggtttctgc cctgaaggct tcctcccctc 2100ccaatgcggt ttaaaacata aataaaaaac cagactctgt ttggatttgg atcaagcaag 2160tgtcttgctg tctttattta ggggttttgc gcgcgcggta ggcccgggac cagcggtctc 2220ggtcgttgag ggtcctgtgt attttttcca ggacgtggta aaggtgactc tggatgttca 2280gatacatggg cataagcccg tctctggggt ggaggtagca ccactgcaga gcttcatgct 2340gcggggtggt gttgtagatg atccagtcgt agcaggagcg ctgggcgtgg tgcctaaaaa 2400tgtctttcag tagcaagctg attgccaggg gcaggccctt ggtgtaagtg tttacaaagc 2460ggttaagctg ggatgggtgc atacgtgggg atatgagatg catcttggac tgtattttta 2520ggttggctat gttcccagcc atatccctcc ggggattcat gttgtgcaga accaccagca 2580cagtgtatcc ggtgcacttg ggaaatttgt catgtagctt agaaggaaat gcgtggaaga 2640acttggagac gcccttgtga cctccaagat tttccatgca ttcgtccata atgatggcaa 2700tgggcccacg ggcggcggcc tgggcgaaga tatttctggg atcactaacg tcatagttgt 2760gttccaggat gagatcgtca taggccattt ttacaaagcg cgggcggagg gtgccagact 2820gcggtataat ggttccatcc ggcccagggg cgtagttacc ctcacagatt tgcatttccc 2880acgctttgag ttcagatggg gggatcatgt ctacctgcgg ggcgatgaag aaaacggttt 2940ccggggtagg ggagatcagc tgggaagaaa gcaggttcct gagcagctgc gacttaccgc 3000agccggtggg cccgtaaatc acacctatta ccgggtgcaa ctggtagtta agagagctgc 3060agctgccgtc atccctgagc aggggggcca cttcgttaag catgtccctg actcgcatgt 3120tttccctgac caaatccgcc agaaggcgct cgccgcccag cgatagcagt tcttgcaagg 3180aagcaaagtt tttcaacggt ttgagaccgt ccgccgtagg catgcttttg agcgtttgac 3240caagcagttc caggcggtcc cacagctcgg tcacctgctc tacggcatct cgatccagca 3300tatctcctcg tttcgcgggt tggggcggct ttcgctgtac ggcagtagtc ggtgctcgtc 3360cagacgggcc agggtcatgt ctttccacgg gcgcagggtc ctcgtcagcg tagtctgggt 3420cacggtgaag gggtgcgctc cgggctgcgc gctggccagg gtgcgcttga ggctggtcct 3480gctggtgctg aagcgctgcc ggtcttcgcc ctgcgcgtcg gccaggtagc atttgaccat 3540ggtgtcatag tccagcccct ccgcggcgtg gcccttggcg cgcagcttgc ccttggagga 3600ggcgccgcac gaggggcagt gcagactttt gagggcgtag agcttgggcg cgagaaatac 3660cgattccggg gagtaggcat ccgcgccgca ggccccgcag acggtctcgc attccacgag 3720ccaggtgagc tctggccgtt cggggtcaaa aaccaggttt cccccatgct ttttgatgcg 3780tttcttacct ctggtttcca tgagccggtg tccacgctcg gtgacgaaaa ggctgtccgt 3840gtccccgtat acagacttga gaggcctgtc ctcgagcggt gttccgcggt cctcctcgta 3900tagaaactcg gaccactctg agacaaaggc tcgcgtccag gccagcacga aggaggctaa 3960gtgggagggg tagcggtcgt tgtccactag ggggtccact cgctccaggg tgtgaagaca 4020catgtcgccc tcttcggcat caaggaaggt gattggtttg taggtgtagg ccacgtgacc 4080gggtgttcct gaaggggggc tataaaaggg ggtgggggcg cgttcgtcct cactctcttc 4140cgcatcgctg tctgcgaggg ccagctgttg gggtgagtac tccctctgaa aagcgggcat 4200gacttctgcg ctaagattgt cagtttccaa aaacgaggag gatttgatat tcacctggcc 4260cgcggtgatg cctttgaggg tggccgcatc catctggtca gaaaagacaa tctttttgtt 4320gtcaagcttg gtggcaaacg acccgtagag ggcgttggac agcaacttgg cgatggagcg 4380cagggtttgg tttttgtcgc gatcggcgcg ctccttggcc gcgatgttta gctgcacgta 4440ttcgcgcgca acgcaccgcc attcgggaaa gacggtggtg cgctcgtcgg gcaccaggtg 4500cacgcgccaa ccgcggttgt gcagggtgac aaggtcaacg ctggtggcta cctctccgcg 4560taggcgctcg ttggtccagc agaggcggcc gcccttgcgc gagcagaatg gcggtagggg 4620gtctagctgc gtctcgtccg gggggtctgc gtccacggta aagaccccgg gcagcaggcg 4680cgcgtcgaag tagtctatct tgcatccttg caagtctagc gcctgctgcc atgcgcgggc 4740ggcaagcgcg cgctcgtatg ggttgagtgg gggaccccat ggcatggggt gggtgagcgc 4800ggaggcgtac atgccgcaaa tgtcgtaaac gtagaggggc tctctgagta ttccaagata 4860tgtagggtag catcttccac cgcggatgct ggcgcgcacg taatcgtata gttcgtgcga 4920gggagcgagg aggtcgggac cgaggttgct acgggcgggc tgctctgctc ggaagactat 4980ctgcctgaag atggcatgtg agttggatga tatggttgga cgctggaaga cgttgaagct 5040ggcgtctgtg agacctaccg cgtcacgcac gaaggaggcg taggagtcgc gcagcttgtt 5100gaccagctcg gcggtgacct gcacgtctag ggcgcagtag tccagggttt ccttgatgat 5160gtcatactta tcctgtccct tttttttcca cagctcgcgg ttgaggacaa actcttcgcg 5220gtctttccag tactcttgga tcggaaaccc gtcggcctcc gaacggtaag agcctagcat 5280gtagaactgg ttgacggcct ggtaggcgca gcatcccttt tctacgggta gcgcgtatgc 5340ctgcgcggcc ttccggagcg aggtgtgggt gagcgcaaag gtgtccctga ccatgacttt 5400gaggtactgg tatttgaagt cagtgtcgtc gcatccgccc tgctcccaga gcaaaaagtc 5460cgtgcgcttt ttggaacgcg gatttggcag ggcgaaggtg acatcgttga agagtatctt 5520tcccgcgcga ggcataaagt tgcgtgtgat gcggaagggt cccggcacct cggaacggtt 5580gttaattacc tgggcggcga gcacgatctc gtcaaagccg ttgatgttgt ggcccacaat 5640gtaaagttcc aagaagcgcg ggatgccctt gatggaaggc aattttttaa gttcctcgta 5700ggtgagctct tcaggggagc tgagcccgtg ctctgaaagg gcccagtctg caagatgagg 5760gttggaagcg acgaatgagc tccacaggtc acgggccatt agcatttgca ggtggtcgcg 5820aaaggtccta aactggcgac ctatggccat tttttctggg gtgatgcagt agaaggtaag 5880cgggtcttgt tcccagcggt cccatccaag gttcgcggct aggtctcgcg cggcagtcac 5940tagaggctca tctccgccga acttcatgac cagcatgaag ggcacgagct gcttcccaaa 6000ggcccccatc caagtatagg tctctacatc gtaggtgaca aagagacgct cggtgcgagg 6060atgcgagccg atcgggaaga actggatctc ccgccaccaa ttggaggagt ggctattgat 6120gtggtgaaag tagaagtccc tgcgacgggc cgaacactcg tgctggcttt tgtaaaaacg 6180tgcgcagtac tggcagcggt gcacgggctg tacatcctgc acgaggttga cctgacgacc 6240gcgcacaagg aagcagagtg ggaatttgag cccctcgcct ggcgggtttg gctggtggtc 6300ttctacttcg gctgcttgtc cttgaccgtc tggctgctcg aggggagtta cggtggatcg 6360gaccaccacg ccgcgcgagc ccaaagtcca gatgtccgcg cgcggcggtc ggagcttgat 6420gacaacatcg cgcagatggg agctgtccat ggtctggagc tcccgcggcg tcaggtcagg 6480cgggagctcc tgcaggttta cctcgcatag acgggtcagg gcgcgggcta gatccaggtg 6540atacctaatt tccaggggct ggttggtggc ggcgtcgatg gcttgcaaga ggccgcatcc 6600ccgcggcgcg actacggtac cgcgcggcgg gcggtgggcc gcgggggtgt ccttggatga 6660tgcatctaaa agcggtgacg cgggcgagcc cccggaggta gggggggctc cggacccgcc 6720gggagagggg gcaggggcac gtcggcgccg cgcgcgggca ggagctggtg ctgcgcgcgt 6780aggttgctgg cgaacgcgac gacgcggcgg ttgatctcct gaatctggcg cctctgcgtg 6840aagacgacgg gcccggtgag cttgagcctg aaagagagtt cgacagaatc aatttcggtg 6900tcgttgacgg cggcctggcg caaaatctcc tgcacgtctc ctgagttgtc ttgataggcg 6960atctcggcca tgaactgctc gatctcttcc tcctggagat ctccgcgtcc ggctcgctcc 7020acggtggcgg cgaggtcgtt ggaaatgcgg gccatgagct gcgagaaggc gttgaggcct 7080ccctcgttcc agacgcggct gtagaccacg cccccttcgg catcgcgggc gcgcatgacc 7140acctgcgcga gattgagctc cacgtgccgg gcgaagacgg cgtagtttcg caggcgctga 7200aagaggtagt tgagggtggt ggcggtgtgt tctgccacga agaagtacat aacccagcgt 7260cgcaacgtgg attcgttgat atcccccaag gcctcaaggc gctccatggc ctcgtagaag 7320tccacggcga agttgaaaaa ctgggagttg cgcgccgaca cggttaactc ctcctccaga 7380agacggatga gctcggcgac agtgtcgcgc acctcgcgct caaaggctac aggggcctct 7440tcttcttctt caatctcctc ttccataagg gcctcccctt cttcttcttc tggcggcggt 7500gggggagggg ggacacggcg gcgacgacgg cgcaccggga ggcggtcgac aaagcgctcg 7560atcatctccc cgcggcgacg gcgcatggtc tcggtgacgg cgcggccgtt ctcgcggggg 7620cgcagttgga agacgccgcc cgtcatgtcc cggttatggg ttggcggggg gctgccatgc 7680ggcagggata cggcgctaac gatgcatctc aacaattgtt gtgtaggtac tccgccgccg 7740agggacctga gcgagtccgc atcgaccgga tcggaaaacc tctcgagaaa ggcgtctaac 7800cagtcacagt cgcaaggtag gctgagcacc gtggcgggcg gcagcgggcg gcggtcgggg 7860ttgtttctgg cggaggtgct gctgatgatg taattaaagt aggcggtctt gagacggcgg 7920atggtcgaca gaagcaccat gtccttgggt ccggcctgct gaatgcgcag gcggtcggcc 7980atgccccagg cttcgttttg acatcggcgc aggtctttgt agtagtcttg catgagcctt 8040tctaccggca cttcttcttc tccttcctct tgtcctgcat ctcttgcatc tatcgctgcg 8100gcggcggcgg agtttggccg taggtggcgc cctcttcctc ccatgcgtgt gaccccgaag 8160cccctcatcg gctgaagcag ggctaggtcg gcgacaacgc gctcggctaa tatggcctgc 8220tgcacctgcg tgagggtaga ctggaagtca tccatgtcca caaagcggtg gtatgcgccc 8280gtgttgatgg tgtaagtgca gttggccata acggaccagt taacggtctg gtgacccggc 8340tgcgagagct cggtgtacct gagacgcgag taagccctcg agtcaaatac gtagtcgttg 8400caagtccgca ccaggtactg gtatcccacc aaaaagtgcg gcggcggctg gcggtagagg 8460ggccagcgta gggtggccgg ggctccgggg gcgagatctt ccaacataag gcgatgatat 8520ccgtagatgt acctggacat ccaggtgatg ccggcggcgg tggtggaggc gcgcggaaag 8580tcgcggacgc ggttccagat gttgcgcagc ggcaaaaagt gctccatggt cgggacgctc 8640tggccggtca ggcgcgcgca atcgttgacg ctctagaccg tgcaaaagga gagcctgtaa 8700gcgggcactc ttccgtggtc tggtggataa attcgcaagg gtatcatggc ggacgaccgg 8760ggttcgagcc ccgtatccgg ccgtccgccg tgatccatgc ggttaccgcc cgcgtgtcga 8820acccaggtgt gcgacgtcag acaacggggg agtgctcctt ttggcttcct tccaggcgcg 8880gcggctgctg cgctagcttt tttggccact ggccgcgcgc agcgtaagcg gttaggctgg 8940aaagcgaaag cattaagtgg ctcgctccct gtagccggag ggttattttc caagggttga 9000gtcgcgggac ccccggttcg agtctcggac cggccggact gcggcgaacg ggggtttgcc 9060tccccgtcat gcaagacccc gcttgcaaat tcctccggaa acagggacga gccccttttt 9120tgcttttccc agatgcatcc ggtgctgcgg cagatgcgcc cccctcctca gcagcggcaa 9180gagcaagagc agcggcagac atgcagggca ccctcccctc ctcctaccgc gtcaggaggg 9240gcgacatccg cggttgacgc ggcagcagat ggtgattacg aacccccgcg gcgccgggcc 9300cggcactacc tggacttgga ggagggcgag ggcctggcgc ggctaggagc gccctctcct 9360gagcggtacc caagggtgca gctgaagcgt gatacgcgtg aggcgtacgt gccgcggcag 9420aacctgtttc gcgaccgcga gggagaggag cccgaggaga tgcgggatcg aaagttccac 9480gcagggcgcg agctgcggca tggcctgaat cgcgagcggt tgctgcgcga ggaggacttt 9540gagcccgacg cgcgaaccgg gattagtccc gcgcgcgcac acgtggcggc cgccgacctg 9600gtaaccgcat acgagcagac ggtgaaccag gagattaact ttcaaaaaag ctttaacaac 9660cacgtgcgta cgcttgtggc gcgcgaggag gtggctatag gactgatgca tctgtgggac 9720tttgtaagcg cgctggagca aaacccaaat agcaagccgc tcatggcgca gctgttcctt 9780atagtgcagc acagcaggga caacgaggca ttcagggatg cgctgctaaa catagtagag 9840cccgagggcc gctggctgct cgatttgata aacatcctgc agagcatagt ggtgcaggag 9900cgcagcttga gcctggctga caaggtggcc gccatcaact attccatgct tagcctgggc 9960aagttttacg cccgcaagat ataccatacc ccttacgttc ccatagacaa ggaggtaaag 10020atcgaggggt tctacatgcg catggcgctg aaggtgctta ccttgagcga cgacctgggc 10080gtttatcgca acgagcgcat ccacaaggcc gtgagcgtga gccggcggcg cgagctcagc 10140gaccgcgagc tgatgcacag cctgcaaagg gccctggctg gcacgggcag cggcgataga 10200gaggccgagt cctactttga cgcgggcgct gacctgcgct gggccccaag ccgacgcgcc 10260ctggaggcag ctggggccgg acctgggctg gcggtggcac ccgcgcgcgc tggcaacgtc 10320ggcggcgtgg aggaatatga cgaggacgat gagtacgagc cagaggacgg cgagtactaa 10380gcggtgatgt ttctgatcag atgatgcaag acgcaacgga cccggcggtg cgggcggcgc 10440tgcagagcca gccgtccggc cttaactcca cggacgactg gcgccaggtc atggaccgca 10500tcatgtcgct gactgcgcgc aatcctgacg cgttccggca gcagccgcag gccaaccggc 10560tctccgcaat tctggaagcg gtggtcccgg cgcgcgcaaa ccccacgcac gagaaggtgc 10620tggcgatcgt aaacgcgctg gccgaaaaca gggccatccg gcccgacgag gccggcctgg 10680tctacgacgc gctgcttcag cgcgtggctc gttacaacag cggcaacgtg cagaccaacc 10740tggaccggct ggtgggggat gtgcgcgagg ccgtggcgca gcgtgagcgc gcgcagcagc 10800agggcaacct gggctccatg gttgcactaa acgccttcct gagtacacag cccgccaacg 10860tgccgcgggg acaggaggac tacaccaact ttgtgagcgc actgcggcta atggtgactg 10920agacaccgca aagtgaggtg taccagtctg ggccagacta ttttttccag accagtagac 10980aaggcctgca gaccgtaaac ctgagccagg ctttcaaaaa cttgcagggg ctgtgggggg 11040tgcgggctcc cacaggcgac cgcgcgaccg tgtctagctt gctgacgccc aactcgcgcc 11100tgttgctgct gctaatagcg cccttcacgg acagtggcag cgtgtcccgg gacacatacc 11160taggtcactt gctgacactg taccgcgagg ccataggtca ggcgcatgtg gacgagcata 11220ctttccagga gattacaagt gtcagccgcg cgctggggca ggaggacacg ggcagcctgg 11280aggcaaccct aaactacctg ctgaccaacc ggcggcagaa gatcccctcg ttgcacagtt 11340taaacagcga ggaggagcgc attttgcgct acgtgcagca gagcgtgagc cttaacctga 11400tgcgcgacgg ggtaacgccc agcgtggcgc tggacatgac cgcgcgcaac atggaaccgg 11460gcatgtatgc ctcaaaccgg ccgtttatca accgcctaat ggactacttg catcgcgcgg 11520ccgccgtgaa ccccgagtat ttcaccaatg ccatcttgaa cccgcactgg ctaccgcccc 11580ctggtttcta caccggggga ttcgaggtgc ccgagggtaa cgatggattc ctctgggacg 11640acatagacga cagcgtgttt tccccgcaac cgcagaccct gctagagttg caacagcgcg 11700agcaggcaga ggcggcgctg cgaaaggaaa gcttccgcag gccaagcagc ttgtccgatc 11760taggcgctgc ggccccgcgg tcagatgcta gtagcccatt tccaagcttg atagggtctc 11820ttaccagcac tcgcaccacc cgcccgcgcc tgctgggcga ggaggagtac ctaaacaact 11880cgctgctgca gccgcagcgc gaaaaaaacc tgcctccggc atttcccaac aacgggatag 11940agagcctagt ggacaagatg agtagatgga agacgtacgc gcaggagcac agggacgtgc 12000caggcccgcg cccgcccacc cgtcgtcaaa ggcacgaccg tcagcggggt ctggtgtggg 12060aggacgatga ctcggcagac gacagcagcg tcctggattt gggagggagt ggcaacccgt 12120ttgcgcacct tcgccccagg ctggggagaa tgttttaaaa aaaaaaaagc atgatgcaaa 12180ataaaaaact caccaaggcc atggcaccga gcgttggttt tcttgtattc cccttagtat 12240gcggcgcgcg gcgatgtatg aggaaggtcc tcctccctcc tacgagagtg tggtgagcgc 12300ggcgccagtg gcggcggcgc tgggttctcc cttcgatgct cccctggacc cgccgtttgt 12360gcctccgcgg tacctgcggc ctaccggggg gagaaacagc atccgttact ctgagttggc 12420acccctattc gacaccaccc gtgtgtacct ggtggacaac aagtcaacgg atgtggcatc 12480cctgaactac cagaacgacc acagcaactt tctgaccacg gtcattcaaa acaatgacta 12540cagcccgggg gaggcaagca cacagaccat caatcttgac gaccggtcgc actggggcgg 12600cgacctgaaa accatcctgc ataccaacat gccaaatgtg aacgagttca tgtttaccaa 12660taagtttaag gcgcgggtga tggtgtcgcg cttgcctact aaggacaatc aggtggagct 12720gaaatacgag tgggtggagt tcacgctgcc cgagggcaac tactccgaga ccatgaccat 12780agaccttatg aacaacgcga tcgtggagca ctacttgaaa gtgggcagac agaacggggt 12840tctggaaagc gacatcgggg taaagtttga cacccgcaac ttcagactgg ggtttgaccc 12900cgtcactggt cttgtcatgc ctggggtata tacaaacgaa gccttccatc cagacatcat 12960tttgctgcca ggatgcgggg tggacttcac ccacagccgc ctgagcaact tgttgggcat 13020ccgcaagcgg caacccttcc aggagggctt taggatcacc tacgatgatc tggagggtgg 13080taacattccc gcactgttgg atgtggacgc ctaccaggcg agcttgaaag atgacaccga 13140acagggcggg ggtggcgcag gcggcagcaa cagcagtggc agcggcgcgg aagagaactc 13200caacgcggca gccgcggcaa tgcagccggt ggaggacatg aacgatcatg ccattcgcgg 13260cgacaccttt gccacacggg ctgaggagaa gcgcgctgag gccgaagcag cggccgaagc 13320tgccgccccc gctgcgcaac ccgaggtcga gaagcctcag aagaaaccgg tgatcaaacc 13380cctgacagag gacagcaaga aacgcagtta caacctaata agcaatgaca gcaccttcac 13440ccagtaccgc agctggtacc ttgcatacaa ctacggcgac cctcagaccg gaatccgctc 13500atggaccctg ctttgcactc ctgacgtaac ctgcggctcg gagcaggtct actggtcgtt 13560gccagacatg atgcaagacc ccgtgacctt ccgctccacg cgccagatca gcaactttcc 13620ggtggtgggc gccgagctgt tgcccgtgca ctccaagagc ttctacaacg accaggccgt 13680ctactcccaa ctcatccgcc agtttacctc tctgacccac gtgttcaatc gctttcccga 13740gaaccagatt ttggcgcgcc cgccagcccc caccatcacc accgtcagtg aaaacgttcc 13800tgctctcaca gatcacggga cgctaccgct gcgcaacagc atcggaggag tccagcgagt 13860gaccattact gacgccagac gccgcacctg cccctacgtt tacaaggccc tgggcatagt 13920ctcgccgcgc gtcctatcga gccgcacttt ttgagcaagc atgtccatcc ttatatcgcc 13980cagcaataac acaggctggg gcctgcgctt cccaagcaag atgtttggcg gggccaagaa 14040gcgctccgac caacacccag tgcgcgtgcg cgggcactac cgcgcgccct ggggcgcgca 14100caaacgcggc cgcactgggc gcaccaccgt cgatgacgcc atcgacgcgg tggtggagga 14160ggcgcgcaac tacacgccca cgccgccacc agtgtccaca gtggacgcgg ccattcagac 14220cgtggtgcgc ggagcccggc gctatgctaa aatgaagaga cggcggaggc gcgtagcacg 14280tcgccaccgc cgccgacccg gcactgccgc ccaacgcgcg gcggcggccc tgcttaaccg 14340cgcacgtcgc accggccgac gggcggccat gcgggccgct cgaaggctgg ccgcgggtat 14400tgtcactgtg ccccccaggt ccaggcgacg agcggccgcc gcagcagccg cggccattag 14460tgctatgact cagggtcgca ggggcaacgt gtattgggtg cgcgactcgg ttagcggcct 14520gcgcgtgccc gtgcgcaccc gccccccgcg caactagatt gcaagaaaaa actacttaga 14580ctcgtactgt tgtatgtatc cagcggcggc ggcgcgcaac gaagctatgt ccaagcgcaa 14640aatcaaagaa gagatgctcc aggtcatcgc gccggagatc tatggccccc cgaagaagga 14700agagcaggat tacaagcccc gaaagctaaa gcgggtcaaa aagaaaaaga aagatgatga 14760tgatgaactt gacgacgagg tggaactgct gcacgctacc gcgcccaggc gacgggtaca 14820gtggaaaggt cgacgcgtaa aacgtgtttt gcgacccggc accaccgtag tctttacgcc 14880cggtgagcgc tccacccgca cctacaagcg cgtgtatgat gaggtgtacg gcgacgagga 14940cctgcttgag caggccaacg agcgcctcgg ggagtttgcc tacggaaagc ggcataagga 15000catgctggcg ttgccgctgg acgagggcaa cccaacacct agcctaaagc ccgtaacact 15060gcagcaggtg ctgcccgcgc ttgcaccgtc cgaagaaaag cgcggcctaa agcgcgagtc 15120tggtgacttg gcacccaccg tgcagctgat ggtacccaag cgccagcgac tggaagatgt 15180cttggaaaaa atgaccgtgg aacctgggct ggagcccgag gtccgcgtgc ggccaatcaa 15240gcaggtggcg ccgggactgg gcgtgcagac cgtggacgtt cagataccca ctaccagtag 15300caccagtatt gccaccgcca cagagggcat ggagacacaa acgtccccgg ttgcctcagc 15360ggtggcggat gccgcggtgc aggcggtcgc tgcggccgcg tccaagacct ctacggaggt 15420gcaaacggac ccgtggatgt ttcgcgtttc agccccccgg cgcccgcgcg gttcgaggaa 15480gtacggcgcc gccagcgcgc tactgcccga atatgcccta catccttcca ttgcgcctac 15540ccccggctat cgtggctaca cctaccgccc cagaagacga gcaactaccc gacgccgaac 15600caccactgga acccgccgcc gccgtcgccg tcgccagccc gtgctggccc cgatttccgt 15660gcgcagggtg gctcgcgaag gaggcaggac cctggtgctg ccaacagcgc gctaccaccc 15720cagcatcgtt taaaagccgg tctttgtggt tcttgcagat atggccctca cctgccgcct 15780ccgtttcccg gtgccgggat tccgaggaag aatgcaccgt aggaggggca tggccggcca 15840cggcctgacg ggcggcatgc gtcgtgcgca ccaccggcgg cggcgcgcgt cgcaccgtcg 15900catgcgcggc ggtatcctgc ccctccttat tccactgatc gccgcggcga ttggcgccgt 15960gcccggaatt gcatccgtgg ccttgcaggc gcagagacac tgattaaaaa caagttgcat 16020gtggaaaaat caaaataaaa agtctggact ctcacgctcg cttggtcctg taactatttt 16080gtagaatgga agacatcaac tttgcgtctc tggccccgcg acacggctcg cgcccgttca 16140tgggaaactg gcaagatatc ggcaccagca atatgagcgg tggcgccttc agctggggct 16200cgctgtggag cggcattaaa aatttcggtt ccaccgttaa gaactatggc agcaaggcct 16260ggaacagcag cacaggccag atgctgaggg ataagttgaa agagcaaaat ttccaacaaa 16320aggtggtaga tggcctggcc tctggcatta gcggggtggt ggacctggcc aaccaggcag 16380tgcaaaataa gattaacagt aagcttgatc cccgccctcc cgtagaggag cctccaccgg 16440ccgtggagac agtgtctcca gaggggcgtg gcgaaaagcg tccgcgcccc gacagggaag 16500aaactctggt gacgcaaata gacgagcctc cctcgtacga ggaggcacta aagcaaggcc 16560tgcccaccac ccgtcccatc gcgcccatgg ctaccggagt gctgggccag cacacacccg 16620taacgctgga cctgcctccc cccgccgaca cccagcagaa acctgtgctg ccaggcccga 16680ccgccgttgt tgtaacccgt cctagccgcg cgtccctgcg ccgcgccgcc agcggtccgc 16740gatcgttgcg gcccgtagcc agtggcaact ggcaaagcac actgaacagc atcgtgggtc 16800tgggggtgca atccctgaag cgccgacgat gcttctgaat agctaacgtg tcgtatgtgt 16860gtcatgtatg cgtccatgtc gccgccagag gagctgctga gccgccgcgc gcccgctttc 16920caagatggct accccttcga tgatgccgca gtggtcttac atgcacatct cgggccagga 16980cgcctcggag tacctgagcc ccgggctggt gcagtttgcc cgcgccaccg agacgtactt 17040cagcctgaat aacaagttta gaaaccccac ggtggcgcct acgcacgacg tgaccacaga 17100ccggtcccag cgtttgacgc tgcggttcat ccctgtggac cgtgaggata ctgcgtactc 17160gtacaaggcg cggttcaccc tagctgtggg tgataaccgt gtgctggaca tggcttccac 17220gtactttgac atccgcggcg tgctggacag gggccctact tttaagccct actctggcac 17280tgcctacaac gccctggctc ccaagggtgc cccaaatcct tgcgaatggg atgaagctgc 17340tactgctctt gaaataaacc tagaagaaga ggacgatgac aacgaagacg aagtagacga 17400gcaagctgag cagcaaaaaa ctcacgtatt tgggcaggcg ccttattctg gtataaatat 17460tacaaaggag ggtattcaaa taggtgtcga aggtcaaaca cctaaatatg ccgataaaac 17520atttcaacct gaacctcaaa taggagaatc tcagtggtac gaaactgaaa ttaatcatgc 17580agctgggaga gtccttaaaa agactacccc aatgaaacca tgttacggtt catatgcaaa 17640acccacaaat gaaaatggag ggcaaggcat tcttgtaaag caacaaaatg gaaagctaga 17700aagtcaagtg gaaatgcaat ttttctcaac tactgaggcg accgcaggca atggtgataa 17760cttgactcct aaagtggtat tgtacagtga agatgtagat atagaaaccc cagacactca 17820tatttcttac atgcccacta ttaaggaagg taactcacga gaactaatgg gccaacaatc 17880tatgcccaac aggcctaatt acattgcttt tagggacaat tttattggtc taatgtatta 17940caacagcacg ggtaatatgg gtgttctggc gggccaagca tcgcagttga atgctgttgt 18000agatttgcaa gacagaaaca cagagctttc ataccagctt ttgcttgatt ccattggtga 18060tagaaccagg tacttttcta tgtggaatca ggctgttgac agctatgatc cagatgttag 18120aattattgaa aatcatggaa ctgaagatga acttccaaat tactgctttc cactgggagg 18180tgtgattaat acagagactc ttaccaaggt aaaacctaaa acaggtcagg aaaatggatg 18240ggaaaaagat gctacagaat tttcagataa aaatgaaata agagttggaa ataattttgc 18300catggaaatc aatctaaatg ccaacctgtg gagaaatttc ctgtactcca acatagcgct 18360gtatttgccc gacaagctaa agtacagtcc ttccaacgta aaaatttctg ataacccaaa 18420cacctacgac tacatgaaca agcgagtggt ggctcccggg ttagtggact gctacattaa 18480ccttggagca cgctggtccc ttgactatat ggacaacgtc aacccattta accaccaccg 18540caatgctggc ctgcgctacc gctcaatgtt gctgggcaat ggtcgctatg tgcccttcca 18600catccaggtg cctcagaagt tctttgccat taaaaacctc cttctcctgc cgggctcata 18660cacctacgag tggaacttca ggaaggatgt taacatggtt ctgcagagct ccctaggaaa 18720tgacctaagg gttgacggag ccagcattaa gtttgatagc atttgccttt acgccacctt 18780cttccccatg gcccacaaca ccgcctccac gcttgaggcc atgcttagaa acgacaccaa 18840cgaccagtcc tttaacgact atctctccgc cgccaacatg ctctacccta tacccgccaa 18900cgctaccaac gtgcccatat ccatcccctc ccgcaactgg gcggctttcc gcggctgggc 18960cttcacgcgc cttaagacta aggaaacccc atcactgggc tcgggctacg acccttatta 19020cacctactct ggctctatac cctacctaga tggaaccttt tacctcaacc acacctttaa 19080gaaggtggcc attacctttg actcttctgt cagctggcct ggcaatgacc gcctgcttac 19140ccccaacgag tttgaaatta agcgctcagt tgacggggag ggttacaacg ttgcccagtg 19200taacatgacc aaagactggt tcctggtaca aatgctagct aactacaaca ttggctacca 19260gggcttctat atcccagaga gctacaagga ccgcatgtac tccttcttta gaaacttcca 19320gcccatgagc cgtcaggtgg tggatgatac taaatacaag gactaccaac aggtgggcat 19380cctacaccaa cacaacaact ctggatttgt tggctacctt gcccccacca tgcgcgaagg 19440acaggcctac cctgctaact tcccctatcc gcttataggc aagaccgcag ttgacagcat 19500tacccagaaa aagtttcttt gcgatcgcac cctttggcgc atcccattct ccagtaactt 19560tatgtccatg ggcgcactca cagacctggg ccaaaacctt ctctacgcca actccgccca 19620cgcgctagac atgacttttg aggtggatcc catggacgag cccacccttc tttatgtttt 19680gtttgaagtc tttgacgtgg tccgtgtgca ccggccgcac cgcggcgtca tcgaaaccgt 19740gtacctgcgc acgcccttct cggccggcaa cgccacaaca taaagaagca agcaacatca 19800acaacagctg ccgccatggg ctccagtgag caggaactga aagccattgt caaagatctt 19860ggttgtgggc catatttttt gggcacctat gacaagcgct ttccaggctt tgtttctcca 19920cacaagctcg cctgcgccat agtcaatacg gccggtcgcg agactggggg cgtacactgg 19980atggcctttg cctggaaccc gcactcaaaa acatgctacc tctttgagcc ctttggcttt 20040tctgaccagc gactcaagca ggtttaccag tttgagtacg agtcactcct gcgccgtagc 20100gccattgctt cttcccccga ccgctgtata acgctggaaa agtccaccca aagcgtacag 20160gggcccaact cggccgcctg tggactattc tgctgcatgt ttctccacgc ctttgccaac 20220tggccccaaa ctcccatgga tcacaacccc accatgaacc ttattaccgg ggtacccaac 20280tccatgctca acagtcccca ggtacagccc accctgcgtc gcaaccagga acagctctac 20340agcttcctgg agcgccactc gccctacttc cgcagccaca gtgcgcagat taggagcgcc 20400acttcttttt gtcacttgaa aaacatgtaa aaataatgta ctagagacac tttcaataaa 20460ggcaaatgct tttatttgta cactctcggg tgattattta cccccaccct tgccgtctgc 20520gccgtttaaa aatcaaaggg gttctgccgc gcatcgctat gcgccactgg cagggacacg 20580ttgcgatact ggtgtttagt gctccactta aactcaggca caaccatccg cggcagctcg 20640gtgaagtttt cactccacag gctgcgcacc atcaccaacg cgtttagcag gtcgggcgcc 20700gatatcttga agtcgcagtt ggggcctccg ccctgcgcgc gcgagttgcg atacacaggg 20760ttgcagcact ggaacactat cagcgccggg tggtgcacgc tggccagcac gctcttgtcg 20820gagatcagat ccgcgtccag gtcctccgcg ttgctcaggg cgaacggagt caactttggt 20880agctgccttc ccaaaaaggg cgcgtgccca ggctttgagt tgcactcgca ccgtagtggc 20940atcaaaaggt gaccgtgccc ggtctgggcg ttaggataca gcgcctgcat aaaagccttg 21000atctgcttaa aagccacctg agcctttgcg ccttcagaga agaacatgcc gcaagacttg 21060ccggaaaact gattggccgg acaggccgcg tcgtgcacgc agcaccttgc gtcggtgttg 21120gagatctgca ccacatttcg gccccaccgg ttcttcacga tcttggcctt gctagactgc 21180tccttcagcg cgcgctgccc gttttcgctc gtcacatcca tttcaatcac gtgctcctta 21240tttatcataa tgcttccgtg tagacactta agctcgcctt cgatctcagc gcagcggtgc 21300agccacaacg cgcagcccgt gggctcgtga tgcttgtagg tcacctctgc aaacgactgc 21360aggtacgcct gcaggaatcg ccccatcatc gtcacaaagg tcttgttgct ggtgaaggtc 21420agctgcaacc cgcggtgctc ctcgttcagc caggtcttgc atacggccgc cagagcttcc 21480acttggtcag gcagtagttt gaagttcgcc tttagatcgt tatccacgtg gtacttgtcc 21540atcagcgcgc gcgcagcctc catgcccttc tcccacgcag acacgatcgg cacactcagc 21600gggttcatca ccgtaatttc actttccgct tcgctgggct cttcctcttc ctcttgcgtc 21660cgcataccac gcgccactgg gtcgtcttca ttcagccgcc gcactgtgcg cttacctcct 21720ttgccatgct tgattagcac cggtgggttg ctgaaaccca ccatttgtag cgccacatct 21780tctctttctt cctcgctgtc cacgattacc tctggtgatg gcgggcgctc gggcttggga 21840gaagggcgct tctttttctt cttgggcgca atggccaaat ccgccgccga ggtcgatggc 21900cgcgggctgg gtgtgcgcgg caccagcgcg tcttgtgatg agtcttcctc gtcctcggac 21960tcgatacgcc gcctcatccg cttttttggg ggcgcccggg gaggcggcgg cgacggggac 22020ggggacgaca cgtcctccat ggttggggga cgtcgcgccg caccgcgtcc gcgctcgggg 22080gtggtttcgc gctgctcctc ttcccgactg gccatttcct tctcctatag gcagaaaaag 22140atcatggagt cagtcgagaa gaaggacagc ctaaccgccc cctctgagtt cgccaccacc 22200gcctccaccg atgccgccaa cgcgcctacc accttccccg tcgaggcacc cccgcttgag 22260gaggaggaag tgattatcga gcaggaccca ggttttgtaa gcgaagacga cgaggaccgc 22320tcagtaccaa cagaggataa aaagcaagac caggacaacg cagaggcaaa cgaggaacaa 22380gtcgggcggg gggacgaaag gcatggcgac tacctagatg tgggagacga cgtgctgttg 22440aagcatctgc agcgccagtg cgccattatc tgcgacgcgt tgcaagagcg cagcgatgtg 22500cccctcgcca tagcggatgt cagccttgcc tacgaacgcc acctattctc accgcgcgta 22560ccccccaaac gccaagaaaa cggcacatgc gagcccaacc cgcgcctcaa cttctacccc 22620gtatttgccg tgccagaggt gcttgccacc tatcacatct ttttccaaaa ctgcaagata 22680cccctatcct gccgtgccaa ccgcagccga gcggacaagc agctggcctt gcggcagggc 22740gctgtcatac ctgatatcgc ctcgctcaac gaagtgccaa aaatctttga gggtcttgga 22800cgcgacgaga agcgcgcggc aaacgctctg caacaggaaa acagcgaaaa tgaaagtcac 22860tctggagtgt tggtggaact cgagggtgac aacgcgcgcc tagccgtact aaaacgcagc 22920atcgaggtca cccactttgc ctacccggca cttaacctac cccccaaggt catgagcaca 22980gtcatgagtg agctgatcgt gcgccgtgcg cagcccctgg agagggatgc aaatttgcaa 23040gaacaaacag aggagggcct acccgcagtt ggcgacgagc agctagcgcg ctggcttcaa 23100acgcgcgagc ctgccgactt ggaggagcga cgcaaactaa tgatggccgc agtgctcgtt 23160accgtggagc ttgagtgcat gcagcggttc tttgctgacc cggagatgca gcgcaagcta 23220gaggaaacat tgcactacac ctttcgacag ggctacgtac gccaggcctg caagatctcc 23280aacgtggagc tctgcaacct ggtctcctac cttggaattt tgcacgaaaa ccgccttggg 23340caaaacgtgc ttcattccac gctcaagggc gaggcgcgcc gcgactacgt ccgcgactgc 23400gtttacttat ttctatgcta cacctggcag acggccatgg gcgtttggca gcagtgcttg 23460gaggagtgca acctcaagga gctgcagaaa ctgctaaagc aaaacttgaa ggacctatgg 23520acggccttca acgagcgctc cgtggccgcg cacctggcgg acatcatttt ccccgaacgc 23580ctgcttaaaa ccctgcaaca gggtctgcca gacttcacca gtcaaagcat gttgcagaac 23640tttaggaact ttatcctaga gcgctcagga atcttgcccg ccacctgctg tgcacttcct 23700agcgactttg tgcccattaa gtaccgcgaa tgccctccgc cgctttgggg ccactgctac 23760cttctgcagc tagccaacta ccttgcctac cactctgaca taatggaaga cgtgagcggt 23820gacggtctac tggagtgtca ctgtcgctgc aacctatgca ccccgcaccg ctccctggtt 23880tgcaattcgc agctgcttaa cgaaagtcaa attatcggta cctttgagct gcagggtccc 23940tcgcctgacg aaaagtccgc ggctccgggg ttgaaactca ctccggggct gtggacgtcg 24000gcttaccttc gcaaatttgt acctgaggac taccacgccc acgagattag gttctacgaa 24060gaccaatccc gcccgccaaa tgcggagctt accgcctgcg tcattaccca gggccacatt 24120cttggccaat tgcaagccat caacaaagcc cgccaagagt ttctgctacg aaagggacgg 24180ggggtttact tggaccccca gtccggcgag gagctcaacc caatcccccc gccgccgcag 24240ccctatcagc agcagccgcg ggcccttgct tcccaggatg gcacccaaaa agaagctgca 24300gctgccgccg ccacccacgg acgaggagga atactgggac agtcaggcag aggaggtttt 24360ggacgaggag gaggaggaca tgatggaaga ctgggagagc ctagacgagg aagcttccga 24420ggtcgaagag gtgtcagacg aaacaccgtc accctcggtc gcattcccct cgccggcgcc 24480ccagaaatcg gcaaccggtt ccagcatggc tacaacctcc gctcctcagg cgccgccggc 24540actgcccgtt cgccgaccca accgtagatg ggacaccact ggaaccaggg ccggtaagtc 24600caagcagccg ccgccgttag cccaagagca acaacagcgc caaggctacc gctcatggcg 24660cgggcacaag aacgccatag ttgcttgctt gcaagactgt gggggcaaca tctccttcgc 24720ccgccgcttt cttctctacc atcacggcgt ggccttcccc cgtaacatcc tgcattacta 24780ccgtcatctc tacagcccat actgcaccgg cggcagcggc agcggcagca acagcagcgg 24840ccacacagaa gcaaaggcga ccggatagca agactctgac aaagcccaag aaatccacag 24900cggcggcagc agcaggagga ggagcgctgc gtctggcgcc caacgaaccc gtatcgaccc 24960gcgagcttag aaacaggatt tttcccactc tgtatgctat atttcaacag agcaggggcc 25020aagaacaaga gctgaaaata aaaaacaggt ctctgcgatc cctcacccgc agctgcctgt 25080atcacaaaag cgaagatcag cttcggcgca cgctggaaga cgcggaggct ctcttcagta 25140aatactgcgc gctgactctt aaggactagt ttcgcgccct ttctcaaatt taagcgcgaa 25200aactacgtca tctccagcgg ccacacccgg cgccagcacc tgtcgtcagc gccattatga 25260gcaaggaaat tcccacgccc tacatgtgga gttaccagcc acaaatggga cttgcggctg 25320gagctgccca agactactca acccgaataa actacatgag cgcgggaccc cacatgatat 25380cccgggtcaa cggaatccgc gcccaccgaa accgaattct cttggaacag gcggctatta 25440ccaccacacc tcgtaataac cttaatcccc gtagttggcc cgctgccctg gtgtaccagg 25500aaagtcccgc tcccaccact gtggtacttc ccagagacgc ccaggccgaa gttcagatga 25560ctaactcagg ggcgcagctt gcgggcggct ttcgtcacag ggtgcggtcg cccgggcagg 25620gtataactca cctgacaatc agagggcgag gtattcagct caacgacgag tcggtgagct 25680cctcgcttgg tctccgtccg gacgggacat ttcagatcgg cggcgccggc cgtccttcat 25740tcacgcctcg tcaggcaatc ctaactctgc agacctcgtc ctctgagccg cgctctggag 25800gcattggaac tctgcaattt attgaggagt ttgtgccatc ggtctacttt aaccccttct 25860cgggacctcc cggccactat ccggatcaat ttattcctaa ctttgacgcg gtaaaggact 25920cggcggacgg ctacgactga atgttaagtg gagaggcaga gcaactgcgc ctgaaacacc 25980tggtccactg tcgccgccac aagtgctttg cccgcgactc cggtgagttt tgctactttg 26040aattgcccga ggatcatatc gagggcccgg cgcacggcgt ccggcttacc gcccagggag 26100agcttgcccg tagcctgatt cgggagttta cccagcgccc cctgctagtt gagcgggaca 26160ggggaccctg tgttctcact gtgatttgca actgtcctaa ccttggatta catcaagatc 26220tttgttgcca tctctgtgct gagtataata aatacagaaa ttaaaatata ctggggctcc 26280tatcgccatc ctgtaaacgc caccgtcttc acccgcccaa gcaaaccaag gcgaacctta 26340cctggtactt ttaacatctc tccctctgtg atttacaaca gtttcaaccc agacggagtg 26400agtctacgag agaacctctc cgagctcagc tactccatca gaaaaaacac caccctcctt 26460acctgccggg aacgtacgag tgcgtcaccg gccgctgcac cacacctacc gcctgaccgt 26520aaaccagact ttttccggac agacctcaat aactctgttt accagaacag gaggtgagct 26580tagaaaaccc ttagggtatt aggccaaagg cgcagctact gtggggttta tgaacaattc 26640aagcaactct acgggctatt ctaattcagg tttctctaga aatggacgga attattacag 26700agcagcgcct gctagaaaga cgcagggcag cggccgagca acagcgcatg aatcaagagc 26760tccaagacat ggttaacttg caccagtgca aaaggggtat cttttgtctg gtaaagcagg 26820ccaaagtcac ctacgacagt aataccaccg gacaccgcct tagctacaag ttgccaacca 26880agcgtcagaa attggtggtc atggtgggag aaaagcccat taccataact cagcactcgg 26940tagaaaccga aggctgcatt cactcacctt gtcaaggacc tgaggatctc tgcaccctta 27000ttaagaccct gtgcggtctc aaagatctta ttccctttaa ctaataaaaa aaaataataa 27060agcatcactt acttaaaatc agttagcaaa tttctgtcca gtttattcag cagcacctcc 27120ttgccctcct cccagctctg gtattgcagc ttcctcctgg ctgcaaactt tctccacaat 27180ctaaatggaa tgtcagtttc ctcctgttcc tgtccatccg cacccactat cttcatgttg 27240ttgcagatga agcgcgcaag accgtctgaa gataccttca accccgtgta tccatatgac 27300acggaaaccg gtcctccaac tgtgcctttt cttactcctc cctttgtatc ccccaatggg 27360tttcaagaga gtccccctgg ggtactctct ttgcgcctat ccgaacctct agttacctcc 27420aatggcatgc ttgcgctcaa aatgggcaac ggcctctctc tggacgaggc cggcaacctt 27480acctcccaaa atgtaaccac tgtgagccca cctctcaaaa aaaccaagtc aaacataaac 27540ctggaaatat ctgcacccct cacagttacc tcagaagccc taactgtggc tgccgccgca 27600cctctaatgg tcgcgggcaa cacactcacc atgcaatcac aggccccgct aaccgtgcac 27660gactccaaac ttagcattgc cacccaagga cccctcacag tgtcagaagg aaagctagcc 27720ctgcaaacat caggccccct caccaccacc gatagcagta cccttactat cactgcctca 27780ccccctctaa ctactgccac tggtagcttg ggcattgact tgaaagagcc catttataca 27840caaaatggaa aactaggact aaagtacggg gctcctttgc atgtaacaga cgacctaaac 27900actttgaccg tagcaactgg tccaggtgtg actattaata atacttcctt gcaaactaaa 27960gttactggag ccttgggttt tgattcacaa ggcaatatgc aacttaatgt agcaggagga 28020ctaaggattg attctcaaaa cagacgcctt atacttgatg ttagttatcc gtttgatgct 28080caaaaccaac taaatctaag actaggacag ggccctcttt ttataaactc agcccacaac 28140ttggatatta actacaacaa aggcctttac ttgtttacag cttcaaacaa ttccaaaaag 28200cttgaggtta acctaagcac tgccaagggg ttgatgtttg acgctacagc catagccatt 28260aatgcaggag atgggcttga atttggttca cctaatgcac caaacacaaa tcccctcaaa 28320acaaaaattg gccatggcct agaatttgat tcaaacaagg ctatggttcc taaactagga 28380actggcctta gttttgacag cacaggtgcc attacagtag gaaacaaaaa taatgataag 28440ctaactttgt ggaccacacc agctccatct cctaactgta gactaaatgc agagaaagat 28500gctaaactca ctttggtctt aacaaaatgt ggcagtcaaa tacttgctac agtttcagtt 28560ttggctgtta aaggcagttt ggctccaata tctggaacag ttcaaagtgc tcatcttatt 28620ataagatttg acgaaaatgg agtgctacta aacaattcct tcctggaccc agaatattgg 28680aactttagaa atggagatct tactgaaggc acagcctata caaacgctgt tggatttatg 28740cctaacctat cagcttatcc aaaatctcac ggtaaaactg ccaaaagtaa cattgtcagt 28800caagtttact taaacggaga caaaactaaa cctgtaacac taaccattac actaaacggt 28860acacaggaaa caggagacac aactccaagt gcatactcta tgtcattttc atgggactgg 28920tctggccaca actacattaa tgaaatattt gccacatcct cttacacttt ttcatacatt 28980gcccaagaat aaagaatcgt ttgtgttatg tttcaacgtg tttatttttc aattgcagaa 29040aatttcgaat catttttcat tcagtagtat agccccacca ccacatagct tatacagatc 29100accgtacctt aatcaaactc acagaaccct agtattcaac ctgccacctc cctcccaaca 29160cacagagtac acagtccttt ctccccggct ggccttaaaa agcatcatat catgggtaac 29220agacatattc ttaggtgtta tattccacac ggtttcctgt cgagccaaac gctcatcagt 29280gatattaata aactccccgg gcagctcact taagttcatg tcgctgtcca gctgctgagc 29340cacaggctgc tgtccaactt gcggttgctt aacgggcggc gaaggagaag tccacgccta 29400catgggggta gagtcataat cgtgcatcag gatagggcgg tggtgctgca gcagcgcgcg 29460aataaactgc tgccgccgcc gctccgtcct gcaggaatac aacatggcag tggtctcctc 29520agcgatgatt cgcaccgccc gcagcataag gcgccttgtc ctccgggcac agcagcgcac 29580cctgatctca cttaaatcag cacagtaact gcagcacagc accacaatat tgttcaaaat 29640cccacagtgc aaggcgctgt atccaaagct catggcgggg accacagaac ccacgtggcc 29700atcataccac aagcgcaggt agattaagtg gcgacccctc ataaacacgc tggacataaa 29760cattacctct tttggcatgt tgtaattcac cacctcccgg taccatataa acctctgatt 29820aaacatggcg ccatccacca ccatcctaaa ccagctggcc aaaacctgcc cgccggctat 29880acactgcagg gaaccgggac tggaacaatg acagtggaga gcccaggact cgtaaccatg 29940gatcatcatg ctcgtcatga tatcaatgtt ggcacaacac aggcacacgt gcatacactt 30000cctcaggatt acaagctcct cccgcgttag aaccatatcc cagggaacaa cccattcctg 30060aatcagcgta aatcccacac tgcagggaag acctcgcacg taactcacgt tgtgcattgt 30120caaagtgtta cattcgggca gcagcggatg atcctccagt atggtagcgc gggtttctgt 30180ctcaaaagga ggtagacgat ccctactgta cggagtgcgc cgagacaacc gagatcgtgt 30240tggtcgtagt gtcatgccaa atggaacgcc ggacgtagtc atatttcctg aagcaaaacc 30300aggtgcgggc gtgacaaaca gatctgcgtc tccggtctcg ccgcttagat cgctctgtgt 30360agtagttgta gtatatccac tctctcaaag catccaggcg ccccctggct tcgggttcta 30420tgtaaactcc ttcatgcgcc gctgccctga taacatccac caccgcagaa taagccacac 30480ccagccaacc tacacattcg ttctgcgagt cacacacggg aggagcggga agagctggaa 30540gaaccatgtt ttttttttta ttccaaaaga ttatccaaaa cctcaaaatg aagatctatt 30600aagtgaacgc gctcccctcc ggtggcgtgg tcaaactcta cagccaaaga acagataatg 30660gcatttgtaa gatgttgcac aatggcttcc aaaaggcaaa cggccctcac gtccaagtgg 30720acgtaaaggc taaacccttc agggtgaatc tcctctataa acattccagc accttcaacc 30780atgcccaaat aattctcatc tcgccacctt ctcaatatat ctctaagcaa atcccgaata 30840ttaagtccgg ccattgtaaa aatctgctcc agagcgccct ccaccttcag cctcaagcag 30900cgaatcatga ttgcaaaaat tcaggttcct cacagacctg tataagattc aaaagcggaa 30960cattaacaaa aataccgcga tcccgtaggt cccttcgcag ggccagctga acataatcgt 31020gcaggtctgc acggaccagc gcggccactt ccccgccagg aaccttgaca aaagaaccca 31080cactgattat gacacgcata ctcggagcta tgctaaccag cgtagccccg atgtaagctt 31140tgttgcatgg gcggcgatat aaaatgcaag gtgctgctca aaaaatcagg caaagcctcg 31200cgcaaaaaag aaagcacatc gtagtcatgc tcatgcagat aaaggcaggt aagctccgga 31260accaccacag aaaaagacac catttttctc tcaaacatgt ctgcgggttt ctgcataaac 31320acaaaataaa ataacaaaaa aacatttaaa cattagaagc ctgtcttaca acaggaaaaa 31380caacccttat aagcataaga cggactacgg ccatgccggc gtgaccgtaa aaaaactggt 31440caccgtgatt aaaaagcacc accgacagct cctcggtcat gtccggagtc ataatgtaag 31500actcggtaaa cacatcaggt tgattcacat cggtcagtgc taaaaagcga ccgaaatagc 31560ccgggggaat acatacccgc aggcgtagag acaacattac agcccccata ggaggtataa 31620caaaattaat aggagagaaa aacacataaa cacctgaaaa accctcctgc ctaggcaaaa 31680tagcaccctc ccgctccaga acaacataca gcgcttccac agcggcagcc ataacagtca 31740gccttaccag taaaaaagaa aacctattaa aaaaacacca ctcgacacgg caccagctca 31800atcagtcaca gtgtaaaaaa gggccaagtg cagagcgagt atatatagga ctaaaaaatg 31860acgtaacggt taaagtccac aaaaaacacc cagaaaaccg cacgcgaacc tacgcccaga 31920aacgaaagcc aaaaaaccca caacttcctc aaatcgtcac ttccgttttc ccacgttacg 31980tcacttccca ttttaagaaa actacaattc ccaacacata caagttactc cgccctaaaa 32040cctacgtcac ccgccccgtt cccacgcccc gcgccacgtc acaaactcca ccccctcatt 32100atcatattgg cttcaatcca aaataaggta tattattgat gatgttaatt aatttaaatc 32160cgcatgcgat atcgagctct cccgggaatt cggatctgcg acgcgaggct ggatggcctt 32220ccccattatg attcttctcg cttccggcgg catcgggatg cccgcgttgc aggccatgct 32280gtccaggcag gtagatgacg accatcaggg acagcttcac ggccagcaaa aggccaggaa 32340ccgtaaaaag gccgcgttgc tggcgttttt ccataggctc cgcccccctg acgagcatca 32400caaaaatcga cgctcaagtc agaggtggcg aaacccgaca ggactataaa gataccaggc 32460gtttccccct ggaagctccc tcgtgcgctc tcctgttccg accctgccgc ttaccggata 32520cctgtccgcc tttctccctt cgggaagcgt ggcgctttct caatgctcac gctgtaggta 32580tctcagttcg gtgtaggtcg ttcgctccaa gctgggctgt gtgcacgaac cccccgttca 32640gcccgaccgc tgcgccttat ccggtaacta tcgtcttgag tccaacccgg taagacacga 32700cttatcgcca ctggcagcag ccactggtaa caggattagc agagcgaggt atgtaggcgg 32760tgctacagag ttcttgaagt ggtggcctaa ctacggctac actagaagga cagtatttgg 32820tatctgcgct ctgctgaagc cagttacctt cggaaaaaga gttggtagct cttgatccgg 32880caaacaaacc accgctggta gcggtggttt ttttgtttgc aagcagcaga ttacgcgcag 32940aaaaaaagga tctcaagaag atcctttgat cttttctacg gggtctgacg ctcagtggaa 33000cgaaaactca cgttaaggga ttttggtcat gagattatca aaaaggatct tcacctagat 33060ccttttaaat caatctaaag tatatatgag taaacttggt ctgacagtta ccaatgctta 33120atcagtgagg cacctatctc agcgatctgt ctatttcgtt catccatagt tgcctgactc 33180cccgtcgtgt agataactac gatacgggag ggcttaccat ctggccccag tgctgcaatg 33240ataccgcgag acccacgctc accggctcca gatttatcag caataaacca gccagccgga 33300agggccgagc gcagaagtgg tcctgcaact ttatccgcct ccatccagtc tattaattgt 33360tgccgggaag ctagagtaag tagttcgcca gttaatagtt tgcgcaacgt tgttgccatt 33420gntgcaggca tcgtggtgtc acgctcgtcg tttggtatgg cttcattcag ctccggttcc 33480caacgatcaa ggcgagttac atgatccccc atgttgtgca aaaaagcggt tagctccttc 33540ggtcctccga tcgttgtcag aagtaagttg gccgcagtgt tatcactcat ggttatggca 33600gcactgcata attctcttac tgtcatgcca tccgtaagat gcttttctgt gactggtgag 33660tactcaacca agtcattctg agaatagtgt atgcggcgac cgagttgctc ttgcccggcg 33720tcaacacggg ataataccgc gccacatagc agaactttaa aagtgctcat cattggaaaa 33780cgttcttcgg ggcgaaaact ctcaaggatc ttaccgctgt tgagatccag ttcgatgtaa 33840cccactcgtg cacccaactg atcttcagca tcttttactt tcaccagcgt ttctgggtga 33900gcaaaaacag gaaggcaaaa tgccgcaaaa aagggaataa gggcgacacg gaaatgttga 33960atactcatac tcttcctttt tcaatattat tgaagcattt atcagggtta ttgtctcatg 34020agcggataca tatttgaatg tatttagaaa aataaacaaa taggggttcc gcgcacattt 34080ccccgaaaag tgccacctga cgtctaagaa accattatta tcatgacatt aacctataaa 34140aataggcgta tcacgaggcc ctttcgtctt caaggatccg aattcccggg agagctcgat 34200atcgcatgcg gatttaaatt aattaa 34226 85 34226 DNA Artificial SequencepAdenoTAG tRNA 85 catcatcaat aatatacctt attttggatt gaagccaata tgataatgagggggtggagt 60 ttgtgacgtg gcgcggggcg tgggaacggg gcgggtgacg tagtagtgtggcggaagtgt 120 gatgttgcaa gtgtggcgga acacatgtaa gcgacggatg tggcaaaagtgacgtttttg 180 gtgtgcgccg gtgtacacag gaagtgacaa ttttcgcgcg gttttaggcggatgttgtag 240 taaatttggg cgtaaccgag taagatttgg ccattttcgc gggaaaactgaataagagga 300 agtgaaatct gaataatttt gtgttactca tagcgcgtaa tatttgtctagggccgcggg 360 gactttgacc gtttacgtgg agactcgccc aggtgttttt ctcaggtgttttccgcgttc 420 cgggtcaaag ttggcgtttt attattatag tcagtcgaag cttggatccggtacctctag 480 aattctcgag cggccgctag cgacatcgat cacaagtttg tacaaaaaagcaggctttaa 540 aggaaccaat tcagtcgact ctagaggatc gaaaccatcc tctgctatatggccgcatat 600 attttacttg aagactagga ccctacagaa aaggggtttt aaagtaggcgtgctaaacgt 660 cagcggacct gacccgtgta agaatccaca aggtatcctg gtggaaatgcgcatttgtag 720 gcttcaatat ctgtaatcct actaattagg tgtggagagc tttcagccagtttcgtaggt 780 ttggagacca tttaggggtt ggcgtgtggc cccctcgtaa agtctttcgtacttcctaca 840 tcagacaagt cttgcaattt gcaatatctc ttttagccaa tatctaaatctttaaaattt 900 tgattttgtt ttttacccag gatgagagac attccagagt tgttaccttgtcaaaataaa 960 caaatttaaa gatgtctgtg aaaagaaaca tatattcctc atgggaatatatccaggttg 1020 ttgaaggagg tacgacctcg agatctctat cactgatagg gagactcgagtgtagtcgtg 1080 gccgagtggt taaggcgatg gactctaaat ccattggggt ctccccgcgcaggttcgaat 1140 cctgccgact acggcgtgct ttttttactc tcgggtagag gaaatccggtgcactacctg 1200 tgcaatcaca cagaataaca tggagtagta ctttttattt tcctgttattatctttctcc 1260 ataaaagtgg aaccagataa ttttagttct tttgtgtaac aagactagagattttttgaa 1320 gtgttacatt ggaaagcact tgaaaacaca agtaatttct gacactgctataaaaatgat 1380 ggaaaaacgc tcaagttgtt ttgcctttca gtcttcttga aatgctgtctccctatctga 1440 aatccagctc acgtctgact tccaaaaccg tgcttgcctt taacttatggaataaatatc 1500 tcaaacagat ccccgggcga gctcgaattc gcggccgcac tcgagatatctagacccagc 1560 tttcttgtac aaagtggtga tcgattcgac agatcactga aatgtgtgggcgtggcttaa 1620 gggtgggaaa gaatatataa ggtgggggtc ttatgtagtt ttgtatctgttttgcagcag 1680 ccgccgccgc catgagcacc aactcgtttg atggaagcat tgtgagctcatatttgacaa 1740 cgcgcatgcc cccatgggcc ggggtgcgtc agaatgtgat gggctccagcattgatggtc 1800 gccccgtcct gcccgcaaac tctactacct tgacctacga gaccgtgtctggaacgccgt 1860 tggagactgc agcctccgcc gccgcttcag ccgctgcagc caccgcccgcgggattgtga 1920 ctgactttgc tttcctgagc ccgcttgcaa gcagtgcagc ttcccgttcatccgcccgcg 1980 atgacaagtt gacggctctt ttggcacaat tggattcttt gacccgggaacttaatgtcg 2040 tttctcagca gctgttggat ctgcgccagc aggtttctgc cctgaaggcttcctcccctc 2100 ccaatgcggt ttaaaacata aataaaaaac cagactctgt ttggatttggatcaagcaag 2160 tgtcttgctg tctttattta ggggttttgc gcgcgcggta ggcccgggaccagcggtctc 2220 ggtcgttgag ggtcctgtgt attttttcca ggacgtggta aaggtgactctggatgttca 2280 gatacatggg cataagcccg tctctggggt ggaggtagca ccactgcagagcttcatgct 2340 gcggggtggt gttgtagatg atccagtcgt agcaggagcg ctgggcgtggtgcctaaaaa 2400 tgtctttcag tagcaagctg attgccaggg gcaggccctt ggtgtaagtgtttacaaagc 2460 ggttaagctg ggatgggtgc atacgtgggg atatgagatg catcttggactgtattttta 2520 ggttggctat gttcccagcc atatccctcc ggggattcat gttgtgcagaaccaccagca 2580 cagtgtatcc ggtgcacttg ggaaatttgt catgtagctt agaaggaaatgcgtggaaga 2640 acttggagac gcccttgtga cctccaagat tttccatgca ttcgtccataatgatggcaa 2700 tgggcccacg ggcggcggcc tgggcgaaga tatttctggg atcactaacgtcatagttgt 2760 gttccaggat gagatcgtca taggccattt ttacaaagcg cgggcggagggtgccagact 2820 gcggtataat ggttccatcc ggcccagggg cgtagttacc ctcacagatttgcatttccc 2880 acgctttgag ttcagatggg gggatcatgt ctacctgcgg ggcgatgaagaaaacggttt 2940 ccggggtagg ggagatcagc tgggaagaaa gcaggttcct gagcagctgcgacttaccgc 3000 agccggtggg cccgtaaatc acacctatta ccgggtgcaa ctggtagttaagagagctgc 3060 agctgccgtc atccctgagc aggggggcca cttcgttaag catgtccctgactcgcatgt 3120 tttccctgac caaatccgcc agaaggcgct cgccgcccag cgatagcagttcttgcaagg 3180 aagcaaagtt tttcaacggt ttgagaccgt ccgccgtagg catgcttttgagcgtttgac 3240 caagcagttc caggcggtcc cacagctcgg tcacctgctc tacggcatctcgatccagca 3300 tatctcctcg tttcgcgggt tggggcggct ttcgctgtac ggcagtagtcggtgctcgtc 3360 cagacgggcc agggtcatgt ctttccacgg gcgcagggtc ctcgtcagcgtagtctgggt 3420 cacggtgaag gggtgcgctc cgggctgcgc gctggccagg gtgcgcttgaggctggtcct 3480 gctggtgctg aagcgctgcc ggtcttcgcc ctgcgcgtcg gccaggtagcatttgaccat 3540 ggtgtcatag tccagcccct ccgcggcgtg gcccttggcg cgcagcttgcccttggagga 3600 ggcgccgcac gaggggcagt gcagactttt gagggcgtag agcttgggcgcgagaaatac 3660 cgattccggg gagtaggcat ccgcgccgca ggccccgcag acggtctcgcattccacgag 3720 ccaggtgagc tctggccgtt cggggtcaaa aaccaggttt cccccatgctttttgatgcg 3780 tttcttacct ctggtttcca tgagccggtg tccacgctcg gtgacgaaaaggctgtccgt 3840 gtccccgtat acagacttga gaggcctgtc ctcgagcggt gttccgcggtcctcctcgta 3900 tagaaactcg gaccactctg agacaaaggc tcgcgtccag gccagcacgaaggaggctaa 3960 gtgggagggg tagcggtcgt tgtccactag ggggtccact cgctccagggtgtgaagaca 4020 catgtcgccc tcttcggcat caaggaaggt gattggtttg taggtgtaggccacgtgacc 4080 gggtgttcct gaaggggggc tataaaaggg ggtgggggcg cgttcgtcctcactctcttc 4140 cgcatcgctg tctgcgaggg ccagctgttg gggtgagtac tccctctgaaaagcgggcat 4200 gacttctgcg ctaagattgt cagtttccaa aaacgaggag gatttgatattcacctggcc 4260 cgcggtgatg cctttgaggg tggccgcatc catctggtca gaaaagacaatctttttgtt 4320 gtcaagcttg gtggcaaacg acccgtagag ggcgttggac agcaacttggcgatggagcg 4380 cagggtttgg tttttgtcgc gatcggcgcg ctccttggcc gcgatgtttagctgcacgta 4440 ttcgcgcgca acgcaccgcc attcgggaaa gacggtggtg cgctcgtcgggcaccaggtg 4500 cacgcgccaa ccgcggttgt gcagggtgac aaggtcaacg ctggtggctacctctccgcg 4560 taggcgctcg ttggtccagc agaggcggcc gcccttgcgc gagcagaatggcggtagggg 4620 gtctagctgc gtctcgtccg gggggtctgc gtccacggta aagaccccgggcagcaggcg 4680 cgcgtcgaag tagtctatct tgcatccttg caagtctagc gcctgctgccatgcgcgggc 4740 ggcaagcgcg cgctcgtatg ggttgagtgg gggaccccat ggcatggggtgggtgagcgc 4800 ggaggcgtac atgccgcaaa tgtcgtaaac gtagaggggc tctctgagtattccaagata 4860 tgtagggtag catcttccac cgcggatgct ggcgcgcacg taatcgtatagttcgtgcga 4920 gggagcgagg aggtcgggac cgaggttgct acgggcgggc tgctctgctcggaagactat 4980 ctgcctgaag atggcatgtg agttggatga tatggttgga cgctggaagacgttgaagct 5040 ggcgtctgtg agacctaccg cgtcacgcac gaaggaggcg taggagtcgcgcagcttgtt 5100 gaccagctcg gcggtgacct gcacgtctag ggcgcagtag tccagggtttccttgatgat 5160 gtcatactta tcctgtccct tttttttcca cagctcgcgg ttgaggacaaactcttcgcg 5220 gtctttccag tactcttgga tcggaaaccc gtcggcctcc gaacggtaagagcctagcat 5280 gtagaactgg ttgacggcct ggtaggcgca gcatcccttt tctacgggtagcgcgtatgc 5340 ctgcgcggcc ttccggagcg aggtgtgggt gagcgcaaag gtgtccctgaccatgacttt 5400 gaggtactgg tatttgaagt cagtgtcgtc gcatccgccc tgctcccagagcaaaaagtc 5460 cgtgcgcttt ttggaacgcg gatttggcag ggcgaaggtg acatcgttgaagagtatctt 5520 tcccgcgcga ggcataaagt tgcgtgtgat gcggaagggt cccggcacctcggaacggtt 5580 gttaattacc tgggcggcga gcacgatctc gtcaaagccg ttgatgttgtggcccacaat 5640 gtaaagttcc aagaagcgcg ggatgccctt gatggaaggc aattttttaagttcctcgta 5700 ggtgagctct tcaggggagc tgagcccgtg ctctgaaagg gcccagtctgcaagatgagg 5760 gttggaagcg acgaatgagc tccacaggtc acgggccatt agcatttgcaggtggtcgcg 5820 aaaggtccta aactggcgac ctatggccat tttttctggg gtgatgcagtagaaggtaag 5880 cgggtcttgt tcccagcggt cccatccaag gttcgcggct aggtctcgcgcggcagtcac 5940 tagaggctca tctccgccga acttcatgac cagcatgaag ggcacgagctgcttcccaaa 6000 ggcccccatc caagtatagg tctctacatc gtaggtgaca aagagacgctcggtgcgagg 6060 atgcgagccg atcgggaaga actggatctc ccgccaccaa ttggaggagtggctattgat 6120 gtggtgaaag tagaagtccc tgcgacgggc cgaacactcg tgctggcttttgtaaaaacg 6180 tgcgcagtac tggcagcggt gcacgggctg tacatcctgc acgaggttgacctgacgacc 6240 gcgcacaagg aagcagagtg ggaatttgag cccctcgcct ggcgggtttggctggtggtc 6300 ttctacttcg gctgcttgtc cttgaccgtc tggctgctcg aggggagttacggtggatcg 6360 gaccaccacg ccgcgcgagc ccaaagtcca gatgtccgcg cgcggcggtcggagcttgat 6420 gacaacatcg cgcagatggg agctgtccat ggtctggagc tcccgcggcgtcaggtcagg 6480 cgggagctcc tgcaggttta cctcgcatag acgggtcagg gcgcgggctagatccaggtg 6540 atacctaatt tccaggggct ggttggtggc ggcgtcgatg gcttgcaagaggccgcatcc 6600 ccgcggcgcg actacggtac cgcgcggcgg gcggtgggcc gcgggggtgtccttggatga 6660 tgcatctaaa agcggtgacg cgggcgagcc cccggaggta gggggggctccggacccgcc 6720 gggagagggg gcaggggcac gtcggcgccg cgcgcgggca ggagctggtgctgcgcgcgt 6780 aggttgctgg cgaacgcgac gacgcggcgg ttgatctcct gaatctggcgcctctgcgtg 6840 aagacgacgg gcccggtgag cttgagcctg aaagagagtt cgacagaatcaatttcggtg 6900 tcgttgacgg cggcctggcg caaaatctcc tgcacgtctc ctgagttgtcttgataggcg 6960 atctcggcca tgaactgctc gatctcttcc tcctggagat ctccgcgtccggctcgctcc 7020 acggtggcgg cgaggtcgtt ggaaatgcgg gccatgagct gcgagaaggcgttgaggcct 7080 ccctcgttcc agacgcggct gtagaccacg cccccttcgg catcgcgggcgcgcatgacc 7140 acctgcgcga gattgagctc cacgtgccgg gcgaagacgg cgtagtttcgcaggcgctga 7200 aagaggtagt tgagggtggt ggcggtgtgt tctgccacga agaagtacataacccagcgt 7260 cgcaacgtgg attcgttgat atcccccaag gcctcaaggc gctccatggcctcgtagaag 7320 tccacggcga agttgaaaaa ctgggagttg cgcgccgaca cggttaactcctcctccaga 7380 agacggatga gctcggcgac agtgtcgcgc acctcgcgct caaaggctacaggggcctct 7440 tcttcttctt caatctcctc ttccataagg gcctcccctt cttcttcttctggcggcggt 7500 gggggagggg ggacacggcg gcgacgacgg cgcaccggga ggcggtcgacaaagcgctcg 7560 atcatctccc cgcggcgacg gcgcatggtc tcggtgacgg cgcggccgttctcgcggggg 7620 cgcagttgga agacgccgcc cgtcatgtcc cggttatggg ttggcggggggctgccatgc 7680 ggcagggata cggcgctaac gatgcatctc aacaattgtt gtgtaggtactccgccgccg 7740 agggacctga gcgagtccgc atcgaccgga tcggaaaacc tctcgagaaaggcgtctaac 7800 cagtcacagt cgcaaggtag gctgagcacc gtggcgggcg gcagcgggcggcggtcgggg 7860 ttgtttctgg cggaggtgct gctgatgatg taattaaagt aggcggtcttgagacggcgg 7920 atggtcgaca gaagcaccat gtccttgggt ccggcctgct gaatgcgcaggcggtcggcc 7980 atgccccagg cttcgttttg acatcggcgc aggtctttgt agtagtcttgcatgagcctt 8040 tctaccggca cttcttcttc tccttcctct tgtcctgcat ctcttgcatctatcgctgcg 8100 gcggcggcgg agtttggccg taggtggcgc cctcttcctc ccatgcgtgtgaccccgaag 8160 cccctcatcg gctgaagcag ggctaggtcg gcgacaacgc gctcggctaatatggcctgc 8220 tgcacctgcg tgagggtaga ctggaagtca tccatgtcca caaagcggtggtatgcgccc 8280 gtgttgatgg tgtaagtgca gttggccata acggaccagt taacggtctggtgacccggc 8340 tgcgagagct cggtgtacct gagacgcgag taagccctcg agtcaaatacgtagtcgttg 8400 caagtccgca ccaggtactg gtatcccacc aaaaagtgcg gcggcggctggcggtagagg 8460 ggccagcgta gggtggccgg ggctccgggg gcgagatctt ccaacataaggcgatgatat 8520 ccgtagatgt acctggacat ccaggtgatg ccggcggcgg tggtggaggcgcgcggaaag 8580 tcgcggacgc ggttccagat gttgcgcagc ggcaaaaagt gctccatggtcgggacgctc 8640 tggccggtca ggcgcgcgca atcgttgacg ctctagaccg tgcaaaaggagagcctgtaa 8700 gcgggcactc ttccgtggtc tggtggataa attcgcaagg gtatcatggcggacgaccgg 8760 ggttcgagcc ccgtatccgg ccgtccgccg tgatccatgc ggttaccgcccgcgtgtcga 8820 acccaggtgt gcgacgtcag acaacggggg agtgctcctt ttggcttccttccaggcgcg 8880 gcggctgctg cgctagcttt tttggccact ggccgcgcgc agcgtaagcggttaggctgg 8940 aaagcgaaag cattaagtgg ctcgctccct gtagccggag ggttattttccaagggttga 9000 gtcgcgggac ccccggttcg agtctcggac cggccggact gcggcgaacgggggtttgcc 9060 tccccgtcat gcaagacccc gcttgcaaat tcctccggaa acagggacgagccccttttt 9120 tgcttttccc agatgcatcc ggtgctgcgg cagatgcgcc cccctcctcagcagcggcaa 9180 gagcaagagc agcggcagac atgcagggca ccctcccctc ctcctaccgcgtcaggaggg 9240 gcgacatccg cggttgacgc ggcagcagat ggtgattacg aacccccgcggcgccgggcc 9300 cggcactacc tggacttgga ggagggcgag ggcctggcgc ggctaggagcgccctctcct 9360 gagcggtacc caagggtgca gctgaagcgt gatacgcgtg aggcgtacgtgccgcggcag 9420 aacctgtttc gcgaccgcga gggagaggag cccgaggaga tgcgggatcgaaagttccac 9480 gcagggcgcg agctgcggca tggcctgaat cgcgagcggt tgctgcgcgaggaggacttt 9540 gagcccgacg cgcgaaccgg gattagtccc gcgcgcgcac acgtggcggccgccgacctg 9600 gtaaccgcat acgagcagac ggtgaaccag gagattaact ttcaaaaaagctttaacaac 9660 cacgtgcgta cgcttgtggc gcgcgaggag gtggctatag gactgatgcatctgtgggac 9720 tttgtaagcg cgctggagca aaacccaaat agcaagccgc tcatggcgcagctgttcctt 9780 atagtgcagc acagcaggga caacgaggca ttcagggatg cgctgctaaacatagtagag 9840 cccgagggcc gctggctgct cgatttgata aacatcctgc agagcatagtggtgcaggag 9900 cgcagcttga gcctggctga caaggtggcc gccatcaact attccatgcttagcctgggc 9960 aagttttacg cccgcaagat ataccatacc ccttacgttc ccatagacaaggaggtaaag 10020 atcgaggggt tctacatgcg catggcgctg aaggtgctta ccttgagcgacgacctgggc 10080 gtttatcgca acgagcgcat ccacaaggcc gtgagcgtga gccggcggcgcgagctcagc 10140 gaccgcgagc tgatgcacag cctgcaaagg gccctggctg gcacgggcagcggcgataga 10200 gaggccgagt cctactttga cgcgggcgct gacctgcgct gggccccaagccgacgcgcc 10260 ctggaggcag ctggggccgg acctgggctg gcggtggcac ccgcgcgcgctggcaacgtc 10320 ggcggcgtgg aggaatatga cgaggacgat gagtacgagc cagaggacggcgagtactaa 10380 gcggtgatgt ttctgatcag atgatgcaag acgcaacgga cccggcggtgcgggcggcgc 10440 tgcagagcca gccgtccggc cttaactcca cggacgactg gcgccaggtcatggaccgca 10500 tcatgtcgct gactgcgcgc aatcctgacg cgttccggca gcagccgcaggccaaccggc 10560 tctccgcaat tctggaagcg gtggtcccgg cgcgcgcaaa ccccacgcacgagaaggtgc 10620 tggcgatcgt aaacgcgctg gccgaaaaca gggccatccg gcccgacgaggccggcctgg 10680 tctacgacgc gctgcttcag cgcgtggctc gttacaacag cggcaacgtgcagaccaacc 10740 tggaccggct ggtgggggat gtgcgcgagg ccgtggcgca gcgtgagcgcgcgcagcagc 10800 agggcaacct gggctccatg gttgcactaa acgccttcct gagtacacagcccgccaacg 10860 tgccgcgggg acaggaggac tacaccaact ttgtgagcgc actgcggctaatggtgactg 10920 agacaccgca aagtgaggtg taccagtctg ggccagacta ttttttccagaccagtagac 10980 aaggcctgca gaccgtaaac ctgagccagg ctttcaaaaa cttgcaggggctgtgggggg 11040 tgcgggctcc cacaggcgac cgcgcgaccg tgtctagctt gctgacgcccaactcgcgcc 11100 tgttgctgct gctaatagcg cccttcacgg acagtggcag cgtgtcccgggacacatacc 11160 taggtcactt gctgacactg taccgcgagg ccataggtca ggcgcatgtggacgagcata 11220 ctttccagga gattacaagt gtcagccgcg cgctggggca ggaggacacgggcagcctgg 11280 aggcaaccct aaactacctg ctgaccaacc ggcggcagaa gatcccctcgttgcacagtt 11340 taaacagcga ggaggagcgc attttgcgct acgtgcagca gagcgtgagccttaacctga 11400 tgcgcgacgg ggtaacgccc agcgtggcgc tggacatgac cgcgcgcaacatggaaccgg 11460 gcatgtatgc ctcaaaccgg ccgtttatca accgcctaat ggactacttgcatcgcgcgg 11520 ccgccgtgaa ccccgagtat ttcaccaatg ccatcttgaa cccgcactggctaccgcccc 11580 ctggtttcta caccggggga ttcgaggtgc ccgagggtaa cgatggattcctctgggacg 11640 acatagacga cagcgtgttt tccccgcaac cgcagaccct gctagagttgcaacagcgcg 11700 agcaggcaga ggcggcgctg cgaaaggaaa gcttccgcag gccaagcagcttgtccgatc 11760 taggcgctgc ggccccgcgg tcagatgcta gtagcccatt tccaagcttgatagggtctc 11820 ttaccagcac tcgcaccacc cgcccgcgcc tgctgggcga ggaggagtacctaaacaact 11880 cgctgctgca gccgcagcgc gaaaaaaacc tgcctccggc atttcccaacaacgggatag 11940 agagcctagt ggacaagatg agtagatgga agacgtacgc gcaggagcacagggacgtgc 12000 caggcccgcg cccgcccacc cgtcgtcaaa ggcacgaccg tcagcggggtctggtgtggg 12060 aggacgatga ctcggcagac gacagcagcg tcctggattt gggagggagtggcaacccgt 12120 ttgcgcacct tcgccccagg ctggggagaa tgttttaaaa aaaaaaaagcatgatgcaaa 12180 ataaaaaact caccaaggcc atggcaccga gcgttggttt tcttgtattccccttagtat 12240 gcggcgcgcg gcgatgtatg aggaaggtcc tcctccctcc tacgagagtgtggtgagcgc 12300 ggcgccagtg gcggcggcgc tgggttctcc cttcgatgct cccctggacccgccgtttgt 12360 gcctccgcgg tacctgcggc ctaccggggg gagaaacagc atccgttactctgagttggc 12420 acccctattc gacaccaccc gtgtgtacct ggtggacaac aagtcaacggatgtggcatc 12480 cctgaactac cagaacgacc acagcaactt tctgaccacg gtcattcaaaacaatgacta 12540 cagcccgggg gaggcaagca cacagaccat caatcttgac gaccggtcgcactggggcgg 12600 cgacctgaaa accatcctgc ataccaacat gccaaatgtg aacgagttcatgtttaccaa 12660 taagtttaag gcgcgggtga tggtgtcgcg cttgcctact aaggacaatcaggtggagct 12720 gaaatacgag tgggtggagt tcacgctgcc cgagggcaac tactccgagaccatgaccat 12780 agaccttatg aacaacgcga tcgtggagca ctacttgaaa gtgggcagacagaacggggt 12840 tctggaaagc gacatcgggg taaagtttga cacccgcaac ttcagactggggtttgaccc 12900 cgtcactggt cttgtcatgc ctggggtata tacaaacgaa gccttccatccagacatcat 12960 tttgctgcca ggatgcgggg tggacttcac ccacagccgc ctgagcaacttgttgggcat 13020 ccgcaagcgg caacccttcc aggagggctt taggatcacc tacgatgatctggagggtgg 13080 taacattccc gcactgttgg atgtggacgc ctaccaggcg agcttgaaagatgacaccga 13140 acagggcggg ggtggcgcag gcggcagcaa cagcagtggc agcggcgcggaagagaactc 13200 caacgcggca gccgcggcaa tgcagccggt ggaggacatg aacgatcatgccattcgcgg 13260 cgacaccttt gccacacggg ctgaggagaa gcgcgctgag gccgaagcagcggccgaagc 13320 tgccgccccc gctgcgcaac ccgaggtcga gaagcctcag aagaaaccggtgatcaaacc 13380 cctgacagag gacagcaaga aacgcagtta caacctaata agcaatgacagcaccttcac 13440 ccagtaccgc agctggtacc ttgcatacaa ctacggcgac cctcagaccggaatccgctc 13500 atggaccctg ctttgcactc ctgacgtaac ctgcggctcg gagcaggtctactggtcgtt 13560 gccagacatg atgcaagacc ccgtgacctt ccgctccacg cgccagatcagcaactttcc 13620 ggtggtgggc gccgagctgt tgcccgtgca ctccaagagc ttctacaacgaccaggccgt 13680 ctactcccaa ctcatccgcc agtttacctc tctgacccac gtgttcaatcgctttcccga 13740 gaaccagatt ttggcgcgcc cgccagcccc caccatcacc accgtcagtgaaaacgttcc 13800 tgctctcaca gatcacggga cgctaccgct gcgcaacagc atcggaggagtccagcgagt 13860 gaccattact gacgccagac gccgcacctg cccctacgtt tacaaggccctgggcatagt 13920 ctcgccgcgc gtcctatcga gccgcacttt ttgagcaagc atgtccatccttatatcgcc 13980 cagcaataac acaggctggg gcctgcgctt cccaagcaag atgtttggcggggccaagaa 14040 gcgctccgac caacacccag tgcgcgtgcg cgggcactac cgcgcgccctggggcgcgca 14100 caaacgcggc cgcactgggc gcaccaccgt cgatgacgcc atcgacgcggtggtggagga 14160 ggcgcgcaac tacacgccca cgccgccacc agtgtccaca gtggacgcggccattcagac 14220 cgtggtgcgc ggagcccggc gctatgctaa aatgaagaga cggcggaggcgcgtagcacg 14280 tcgccaccgc cgccgacccg gcactgccgc ccaacgcgcg gcggcggccctgcttaaccg 14340 cgcacgtcgc accggccgac gggcggccat gcgggccgct cgaaggctggccgcgggtat 14400 tgtcactgtg ccccccaggt ccaggcgacg agcggccgcc gcagcagccgcggccattag 14460 tgctatgact cagggtcgca ggggcaacgt gtattgggtg cgcgactcggttagcggcct 14520 gcgcgtgccc gtgcgcaccc gccccccgcg caactagatt gcaagaaaaaactacttaga 14580 ctcgtactgt tgtatgtatc cagcggcggc ggcgcgcaac gaagctatgtccaagcgcaa 14640 aatcaaagaa gagatgctcc aggtcatcgc gccggagatc tatggccccccgaagaagga 14700 agagcaggat tacaagcccc gaaagctaaa gcgggtcaaa aagaaaaagaaagatgatga 14760 tgatgaactt gacgacgagg tggaactgct gcacgctacc gcgcccaggcgacgggtaca 14820 gtggaaaggt cgacgcgtaa aacgtgtttt gcgacccggc accaccgtagtctttacgcc 14880 cggtgagcgc tccacccgca cctacaagcg cgtgtatgat gaggtgtacggcgacgagga 14940 cctgcttgag caggccaacg agcgcctcgg ggagtttgcc tacggaaagcggcataagga 15000 catgctggcg ttgccgctgg acgagggcaa cccaacacct agcctaaagcccgtaacact 15060 gcagcaggtg ctgcccgcgc ttgcaccgtc cgaagaaaag cgcggcctaaagcgcgagtc 15120 tggtgacttg gcacccaccg tgcagctgat ggtacccaag cgccagcgactggaagatgt 15180 cttggaaaaa atgaccgtgg aacctgggct ggagcccgag gtccgcgtgcggccaatcaa 15240 gcaggtggcg ccgggactgg gcgtgcagac cgtggacgtt cagatacccactaccagtag 15300 caccagtatt gccaccgcca cagagggcat ggagacacaa acgtccccggttgcctcagc 15360 ggtggcggat gccgcggtgc aggcggtcgc tgcggccgcg tccaagacctctacggaggt 15420 gcaaacggac ccgtggatgt ttcgcgtttc agccccccgg cgcccgcgcggttcgaggaa 15480 gtacggcgcc gccagcgcgc tactgcccga atatgcccta catccttccattgcgcctac 15540 ccccggctat cgtggctaca cctaccgccc cagaagacga gcaactacccgacgccgaac 15600 caccactgga acccgccgcc gccgtcgccg tcgccagccc gtgctggccccgatttccgt 15660 gcgcagggtg gctcgcgaag gaggcaggac cctggtgctg ccaacagcgcgctaccaccc 15720 cagcatcgtt taaaagccgg tctttgtggt tcttgcagat atggccctcacctgccgcct 15780 ccgtttcccg gtgccgggat tccgaggaag aatgcaccgt aggaggggcatggccggcca 15840 cggcctgacg ggcggcatgc gtcgtgcgca ccaccggcgg cggcgcgcgtcgcaccgtcg 15900 catgcgcggc ggtatcctgc ccctccttat tccactgatc gccgcggcgattggcgccgt 15960 gcccggaatt gcatccgtgg ccttgcaggc gcagagacac tgattaaaaacaagttgcat 16020 gtggaaaaat caaaataaaa agtctggact ctcacgctcg cttggtcctgtaactatttt 16080 gtagaatgga agacatcaac tttgcgtctc tggccccgcg acacggctcgcgcccgttca 16140 tgggaaactg gcaagatatc ggcaccagca atatgagcgg tggcgccttcagctggggct 16200 cgctgtggag cggcattaaa aatttcggtt ccaccgttaa gaactatggcagcaaggcct 16260 ggaacagcag cacaggccag atgctgaggg ataagttgaa agagcaaaatttccaacaaa 16320 aggtggtaga tggcctggcc tctggcatta gcggggtggt ggacctggccaaccaggcag 16380 tgcaaaataa gattaacagt aagcttgatc cccgccctcc cgtagaggagcctccaccgg 16440 ccgtggagac agtgtctcca gaggggcgtg gcgaaaagcg tccgcgccccgacagggaag 16500 aaactctggt gacgcaaata gacgagcctc cctcgtacga ggaggcactaaagcaaggcc 16560 tgcccaccac ccgtcccatc gcgcccatgg ctaccggagt gctgggccagcacacacccg 16620 taacgctgga cctgcctccc cccgccgaca cccagcagaa acctgtgctgccaggcccga 16680 ccgccgttgt tgtaacccgt cctagccgcg cgtccctgcg ccgcgccgccagcggtccgc 16740 gatcgttgcg gcccgtagcc agtggcaact ggcaaagcac actgaacagcatcgtgggtc 16800 tgggggtgca atccctgaag cgccgacgat gcttctgaat agctaacgtgtcgtatgtgt 16860 gtcatgtatg cgtccatgtc gccgccagag gagctgctga gccgccgcgcgcccgctttc 16920 caagatggct accccttcga tgatgccgca gtggtcttac atgcacatctcgggccagga 16980 cgcctcggag tacctgagcc ccgggctggt gcagtttgcc cgcgccaccgagacgtactt 17040 cagcctgaat aacaagttta gaaaccccac ggtggcgcct acgcacgacgtgaccacaga 17100 ccggtcccag cgtttgacgc tgcggttcat ccctgtggac cgtgaggatactgcgtactc 17160 gtacaaggcg cggttcaccc tagctgtggg tgataaccgt gtgctggacatggcttccac 17220 gtactttgac atccgcggcg tgctggacag gggccctact tttaagccctactctggcac 17280 tgcctacaac gccctggctc ccaagggtgc cccaaatcct tgcgaatgggatgaagctgc 17340 tactgctctt gaaataaacc tagaagaaga ggacgatgac aacgaagacgaagtagacga 17400 gcaagctgag cagcaaaaaa ctcacgtatt tgggcaggcg ccttattctggtataaatat 17460 tacaaaggag ggtattcaaa taggtgtcga aggtcaaaca cctaaatatgccgataaaac 17520 atttcaacct gaacctcaaa taggagaatc tcagtggtac gaaactgaaattaatcatgc 17580 agctgggaga gtccttaaaa agactacccc aatgaaacca tgttacggttcatatgcaaa 17640 acccacaaat gaaaatggag ggcaaggcat tcttgtaaag caacaaaatggaaagctaga 17700 aagtcaagtg gaaatgcaat ttttctcaac tactgaggcg accgcaggcaatggtgataa 17760 cttgactcct aaagtggtat tgtacagtga agatgtagat atagaaaccccagacactca 17820 tatttcttac atgcccacta ttaaggaagg taactcacga gaactaatgggccaacaatc 17880 tatgcccaac aggcctaatt acattgcttt tagggacaat tttattggtctaatgtatta 17940 caacagcacg ggtaatatgg gtgttctggc gggccaagca tcgcagttgaatgctgttgt 18000 agatttgcaa gacagaaaca cagagctttc ataccagctt ttgcttgattccattggtga 18060 tagaaccagg tacttttcta tgtggaatca ggctgttgac agctatgatccagatgttag 18120 aattattgaa aatcatggaa ctgaagatga acttccaaat tactgctttccactgggagg 18180 tgtgattaat acagagactc ttaccaaggt aaaacctaaa acaggtcaggaaaatggatg 18240 ggaaaaagat gctacagaat tttcagataa aaatgaaata agagttggaaataattttgc 18300 catggaaatc aatctaaatg ccaacctgtg gagaaatttc ctgtactccaacatagcgct 18360 gtatttgccc gacaagctaa agtacagtcc ttccaacgta aaaatttctgataacccaaa 18420 cacctacgac tacatgaaca agcgagtggt ggctcccggg ttagtggactgctacattaa 18480 ccttggagca cgctggtccc ttgactatat ggacaacgtc aacccatttaaccaccaccg 18540 caatgctggc ctgcgctacc gctcaatgtt gctgggcaat ggtcgctatgtgcccttcca 18600 catccaggtg cctcagaagt tctttgccat taaaaacctc cttctcctgccgggctcata 18660 cacctacgag tggaacttca ggaaggatgt taacatggtt ctgcagagctccctaggaaa 18720 tgacctaagg gttgacggag ccagcattaa gtttgatagc atttgcctttacgccacctt 18780 cttccccatg gcccacaaca ccgcctccac gcttgaggcc atgcttagaaacgacaccaa 18840 cgaccagtcc tttaacgact atctctccgc cgccaacatg ctctaccctatacccgccaa 18900 cgctaccaac gtgcccatat ccatcccctc ccgcaactgg gcggctttccgcggctgggc 18960 cttcacgcgc cttaagacta aggaaacccc atcactgggc tcgggctacgacccttatta 19020 cacctactct ggctctatac cctacctaga tggaaccttt tacctcaaccacacctttaa 19080 gaaggtggcc attacctttg actcttctgt cagctggcct ggcaatgaccgcctgcttac 19140 ccccaacgag tttgaaatta agcgctcagt tgacggggag ggttacaacgttgcccagtg 19200 taacatgacc aaagactggt tcctggtaca aatgctagct aactacaacattggctacca 19260 gggcttctat atcccagaga gctacaagga ccgcatgtac tccttctttagaaacttcca 19320 gcccatgagc cgtcaggtgg tggatgatac taaatacaag gactaccaacaggtgggcat 19380 cctacaccaa cacaacaact ctggatttgt tggctacctt gcccccaccatgcgcgaagg 19440 acaggcctac cctgctaact tcccctatcc gcttataggc aagaccgcagttgacagcat 19500 tacccagaaa aagtttcttt gcgatcgcac cctttggcgc atcccattctccagtaactt 19560 tatgtccatg ggcgcactca cagacctggg ccaaaacctt ctctacgccaactccgccca 19620 cgcgctagac atgacttttg aggtggatcc catggacgag cccacccttctttatgtttt 19680 gtttgaagtc tttgacgtgg tccgtgtgca ccggccgcac cgcggcgtcatcgaaaccgt 19740 gtacctgcgc acgcccttct cggccggcaa cgccacaaca taaagaagcaagcaacatca 19800 acaacagctg ccgccatggg ctccagtgag caggaactga aagccattgtcaaagatctt 19860 ggttgtgggc catatttttt gggcacctat gacaagcgct ttccaggctttgtttctcca 19920 cacaagctcg cctgcgccat agtcaatacg gccggtcgcg agactgggggcgtacactgg 19980 atggcctttg cctggaaccc gcactcaaaa acatgctacc tctttgagccctttggcttt 20040 tctgaccagc gactcaagca ggtttaccag tttgagtacg agtcactcctgcgccgtagc 20100 gccattgctt cttcccccga ccgctgtata acgctggaaa agtccacccaaagcgtacag 20160 gggcccaact cggccgcctg tggactattc tgctgcatgt ttctccacgcctttgccaac 20220 tggccccaaa ctcccatgga tcacaacccc accatgaacc ttattaccggggtacccaac 20280 tccatgctca acagtcccca ggtacagccc accctgcgtc gcaaccaggaacagctctac 20340 agcttcctgg agcgccactc gccctacttc cgcagccaca gtgcgcagattaggagcgcc 20400 acttcttttt gtcacttgaa aaacatgtaa aaataatgta ctagagacactttcaataaa 20460 ggcaaatgct tttatttgta cactctcggg tgattattta cccccacccttgccgtctgc 20520 gccgtttaaa aatcaaaggg gttctgccgc gcatcgctat gcgccactggcagggacacg 20580 ttgcgatact ggtgtttagt gctccactta aactcaggca caaccatccgcggcagctcg 20640 gtgaagtttt cactccacag gctgcgcacc atcaccaacg cgtttagcaggtcgggcgcc 20700 gatatcttga agtcgcagtt ggggcctccg ccctgcgcgc gcgagttgcgatacacaggg 20760 ttgcagcact ggaacactat cagcgccggg tggtgcacgc tggccagcacgctcttgtcg 20820 gagatcagat ccgcgtccag gtcctccgcg ttgctcaggg cgaacggagtcaactttggt 20880 agctgccttc ccaaaaaggg cgcgtgccca ggctttgagt tgcactcgcaccgtagtggc 20940 atcaaaaggt gaccgtgccc ggtctgggcg ttaggataca gcgcctgcataaaagccttg 21000 atctgcttaa aagccacctg agcctttgcg ccttcagaga agaacatgccgcaagacttg 21060 ccggaaaact gattggccgg acaggccgcg tcgtgcacgc agcaccttgcgtcggtgttg 21120 gagatctgca ccacatttcg gccccaccgg ttcttcacga tcttggccttgctagactgc 21180 tccttcagcg cgcgctgccc gttttcgctc gtcacatcca tttcaatcacgtgctcctta 21240 tttatcataa tgcttccgtg tagacactta agctcgcctt cgatctcagcgcagcggtgc 21300 agccacaacg cgcagcccgt gggctcgtga tgcttgtagg tcacctctgcaaacgactgc 21360 aggtacgcct gcaggaatcg ccccatcatc gtcacaaagg tcttgttgctggtgaaggtc 21420 agctgcaacc cgcggtgctc ctcgttcagc caggtcttgc atacggccgccagagcttcc 21480 acttggtcag gcagtagttt gaagttcgcc tttagatcgt tatccacgtggtacttgtcc 21540 atcagcgcgc gcgcagcctc catgcccttc tcccacgcag acacgatcggcacactcagc 21600 gggttcatca ccgtaatttc actttccgct tcgctgggct cttcctcttcctcttgcgtc 21660 cgcataccac gcgccactgg gtcgtcttca ttcagccgcc gcactgtgcgcttacctcct 21720 ttgccatgct tgattagcac cggtgggttg ctgaaaccca ccatttgtagcgccacatct 21780 tctctttctt cctcgctgtc cacgattacc tctggtgatg gcgggcgctcgggcttggga 21840 gaagggcgct tctttttctt cttgggcgca atggccaaat ccgccgccgaggtcgatggc 21900 cgcgggctgg gtgtgcgcgg caccagcgcg tcttgtgatg agtcttcctcgtcctcggac 21960 tcgatacgcc gcctcatccg cttttttggg ggcgcccggg gaggcggcggcgacggggac 22020 ggggacgaca cgtcctccat ggttggggga cgtcgcgccg caccgcgtccgcgctcgggg 22080 gtggtttcgc gctgctcctc ttcccgactg gccatttcct tctcctataggcagaaaaag 22140 atcatggagt cagtcgagaa gaaggacagc ctaaccgccc cctctgagttcgccaccacc 22200 gcctccaccg atgccgccaa cgcgcctacc accttccccg tcgaggcacccccgcttgag 22260 gaggaggaag tgattatcga gcaggaccca ggttttgtaa gcgaagacgacgaggaccgc 22320 tcagtaccaa cagaggataa aaagcaagac caggacaacg cagaggcaaacgaggaacaa 22380 gtcgggcggg gggacgaaag gcatggcgac tacctagatg tgggagacgacgtgctgttg 22440 aagcatctgc agcgccagtg cgccattatc tgcgacgcgt tgcaagagcgcagcgatgtg 22500 cccctcgcca tagcggatgt cagccttgcc tacgaacgcc acctattctcaccgcgcgta 22560 ccccccaaac gccaagaaaa cggcacatgc gagcccaacc cgcgcctcaacttctacccc 22620 gtatttgccg tgccagaggt gcttgccacc tatcacatct ttttccaaaactgcaagata 22680 cccctatcct gccgtgccaa ccgcagccga gcggacaagc agctggccttgcggcagggc 22740 gctgtcatac ctgatatcgc ctcgctcaac gaagtgccaa aaatctttgagggtcttgga 22800 cgcgacgaga agcgcgcggc aaacgctctg caacaggaaa acagcgaaaatgaaagtcac 22860 tctggagtgt tggtggaact cgagggtgac aacgcgcgcc tagccgtactaaaacgcagc 22920 atcgaggtca cccactttgc ctacccggca cttaacctac cccccaaggtcatgagcaca 22980 gtcatgagtg agctgatcgt gcgccgtgcg cagcccctgg agagggatgcaaatttgcaa 23040 gaacaaacag aggagggcct acccgcagtt ggcgacgagc agctagcgcgctggcttcaa 23100 acgcgcgagc ctgccgactt ggaggagcga cgcaaactaa tgatggccgcagtgctcgtt 23160 accgtggagc ttgagtgcat gcagcggttc tttgctgacc cggagatgcagcgcaagcta 23220 gaggaaacat tgcactacac ctttcgacag ggctacgtac gccaggcctgcaagatctcc 23280 aacgtggagc tctgcaacct ggtctcctac cttggaattt tgcacgaaaaccgccttggg 23340 caaaacgtgc ttcattccac gctcaagggc gaggcgcgcc gcgactacgtccgcgactgc 23400 gtttacttat ttctatgcta cacctggcag acggccatgg gcgtttggcagcagtgcttg 23460 gaggagtgca acctcaagga gctgcagaaa ctgctaaagc aaaacttgaaggacctatgg 23520 acggccttca acgagcgctc cgtggccgcg cacctggcgg acatcattttccccgaacgc 23580 ctgcttaaaa ccctgcaaca gggtctgcca gacttcacca gtcaaagcatgttgcagaac 23640 tttaggaact ttatcctaga gcgctcagga atcttgcccg ccacctgctgtgcacttcct 23700 agcgactttg tgcccattaa gtaccgcgaa tgccctccgc cgctttggggccactgctac 23760 cttctgcagc tagccaacta ccttgcctac cactctgaca taatggaagacgtgagcggt 23820 gacggtctac tggagtgtca ctgtcgctgc aacctatgca ccccgcaccgctccctggtt 23880 tgcaattcgc agctgcttaa cgaaagtcaa attatcggta cctttgagctgcagggtccc 23940 tcgcctgacg aaaagtccgc ggctccgggg ttgaaactca ctccggggctgtggacgtcg 24000 gcttaccttc gcaaatttgt acctgaggac taccacgccc acgagattaggttctacgaa 24060 gaccaatccc gcccgccaaa tgcggagctt accgcctgcg tcattacccagggccacatt 24120 cttggccaat tgcaagccat caacaaagcc cgccaagagt ttctgctacgaaagggacgg 24180 ggggtttact tggaccccca gtccggcgag gagctcaacc caatccccccgccgccgcag 24240 ccctatcagc agcagccgcg ggcccttgct tcccaggatg gcacccaaaaagaagctgca 24300 gctgccgccg ccacccacgg acgaggagga atactgggac agtcaggcagaggaggtttt 24360 ggacgaggag gaggaggaca tgatggaaga ctgggagagc ctagacgaggaagcttccga 24420 ggtcgaagag gtgtcagacg aaacaccgtc accctcggtc gcattcccctcgccggcgcc 24480 ccagaaatcg gcaaccggtt ccagcatggc tacaacctcc gctcctcaggcgccgccggc 24540 actgcccgtt cgccgaccca accgtagatg ggacaccact ggaaccagggccggtaagtc 24600 caagcagccg ccgccgttag cccaagagca acaacagcgc caaggctaccgctcatggcg 24660 cgggcacaag aacgccatag ttgcttgctt gcaagactgt gggggcaacatctccttcgc 24720 ccgccgcttt cttctctacc atcacggcgt ggccttcccc cgtaacatcctgcattacta 24780 ccgtcatctc tacagcccat actgcaccgg cggcagcggc agcggcagcaacagcagcgg 24840 ccacacagaa gcaaaggcga ccggatagca agactctgac aaagcccaagaaatccacag 24900 cggcggcagc agcaggagga ggagcgctgc gtctggcgcc caacgaacccgtatcgaccc 24960 gcgagcttag aaacaggatt tttcccactc tgtatgctat atttcaacagagcaggggcc 25020 aagaacaaga gctgaaaata aaaaacaggt ctctgcgatc cctcacccgcagctgcctgt 25080 atcacaaaag cgaagatcag cttcggcgca cgctggaaga cgcggaggctctcttcagta 25140 aatactgcgc gctgactctt aaggactagt ttcgcgccct ttctcaaatttaagcgcgaa 25200 aactacgtca tctccagcgg ccacacccgg cgccagcacc tgtcgtcagcgccattatga 25260 gcaaggaaat tcccacgccc tacatgtgga gttaccagcc acaaatgggacttgcggctg 25320 gagctgccca agactactca acccgaataa actacatgag cgcgggaccccacatgatat 25380 cccgggtcaa cggaatccgc gcccaccgaa accgaattct cttggaacaggcggctatta 25440 ccaccacacc tcgtaataac cttaatcccc gtagttggcc cgctgccctggtgtaccagg 25500 aaagtcccgc tcccaccact gtggtacttc ccagagacgc ccaggccgaagttcagatga 25560 ctaactcagg ggcgcagctt gcgggcggct ttcgtcacag ggtgcggtcgcccgggcagg 25620 gtataactca cctgacaatc agagggcgag gtattcagct caacgacgagtcggtgagct 25680 cctcgcttgg tctccgtccg gacgggacat ttcagatcgg cggcgccggccgtccttcat 25740 tcacgcctcg tcaggcaatc ctaactctgc agacctcgtc ctctgagccgcgctctggag 25800 gcattggaac tctgcaattt attgaggagt ttgtgccatc ggtctactttaaccccttct 25860 cgggacctcc cggccactat ccggatcaat ttattcctaa ctttgacgcggtaaaggact 25920 cggcggacgg ctacgactga atgttaagtg gagaggcaga gcaactgcgcctgaaacacc 25980 tggtccactg tcgccgccac aagtgctttg cccgcgactc cggtgagttttgctactttg 26040 aattgcccga ggatcatatc gagggcccgg cgcacggcgt ccggcttaccgcccagggag 26100 agcttgcccg tagcctgatt cgggagttta cccagcgccc cctgctagttgagcgggaca 26160 ggggaccctg tgttctcact gtgatttgca actgtcctaa ccttggattacatcaagatc 26220 tttgttgcca tctctgtgct gagtataata aatacagaaa ttaaaatatactggggctcc 26280 tatcgccatc ctgtaaacgc caccgtcttc acccgcccaa gcaaaccaaggcgaacctta 26340 cctggtactt ttaacatctc tccctctgtg atttacaaca gtttcaacccagacggagtg 26400 agtctacgag agaacctctc cgagctcagc tactccatca gaaaaaacaccaccctcctt 26460 acctgccggg aacgtacgag tgcgtcaccg gccgctgcac cacacctaccgcctgaccgt 26520 aaaccagact ttttccggac agacctcaat aactctgttt accagaacaggaggtgagct 26580 tagaaaaccc ttagggtatt aggccaaagg cgcagctact gtggggtttatgaacaattc 26640 aagcaactct acgggctatt ctaattcagg tttctctaga aatggacggaattattacag 26700 agcagcgcct gctagaaaga cgcagggcag cggccgagca acagcgcatgaatcaagagc 26760 tccaagacat ggttaacttg caccagtgca aaaggggtat cttttgtctggtaaagcagg 26820 ccaaagtcac ctacgacagt aataccaccg gacaccgcct tagctacaagttgccaacca 26880 agcgtcagaa attggtggtc atggtgggag aaaagcccat taccataactcagcactcgg 26940 tagaaaccga aggctgcatt cactcacctt gtcaaggacc tgaggatctctgcaccctta 27000 ttaagaccct gtgcggtctc aaagatctta ttccctttaa ctaataaaaaaaaataataa 27060 agcatcactt acttaaaatc agttagcaaa tttctgtcca gtttattcagcagcacctcc 27120 ttgccctcct cccagctctg gtattgcagc ttcctcctgg ctgcaaactttctccacaat 27180 ctaaatggaa tgtcagtttc ctcctgttcc tgtccatccg cacccactatcttcatgttg 27240 ttgcagatga agcgcgcaag accgtctgaa gataccttca accccgtgtatccatatgac 27300 acggaaaccg gtcctccaac tgtgcctttt cttactcctc cctttgtatcccccaatggg 27360 tttcaagaga gtccccctgg ggtactctct ttgcgcctat ccgaacctctagttacctcc 27420 aatggcatgc ttgcgctcaa aatgggcaac ggcctctctc tggacgaggccggcaacctt 27480 acctcccaaa atgtaaccac tgtgagccca cctctcaaaa aaaccaagtcaaacataaac 27540 ctggaaatat ctgcacccct cacagttacc tcagaagccc taactgtggctgccgccgca 27600 cctctaatgg tcgcgggcaa cacactcacc atgcaatcac aggccccgctaaccgtgcac 27660 gactccaaac ttagcattgc cacccaagga cccctcacag tgtcagaaggaaagctagcc 27720 ctgcaaacat caggccccct caccaccacc gatagcagta cccttactatcactgcctca 27780 ccccctctaa ctactgccac tggtagcttg ggcattgact tgaaagagcccatttataca 27840 caaaatggaa aactaggact aaagtacggg gctcctttgc atgtaacagacgacctaaac 27900 actttgaccg tagcaactgg tccaggtgtg actattaata atacttccttgcaaactaaa 27960 gttactggag ccttgggttt tgattcacaa ggcaatatgc aacttaatgtagcaggagga 28020 ctaaggattg attctcaaaa cagacgcctt atacttgatg ttagttatccgtttgatgct 28080 caaaaccaac taaatctaag actaggacag ggccctcttt ttataaactcagcccacaac 28140 ttggatatta actacaacaa aggcctttac ttgtttacag cttcaaacaattccaaaaag 28200 cttgaggtta acctaagcac tgccaagggg ttgatgtttg acgctacagccatagccatt 28260 aatgcaggag atgggcttga atttggttca cctaatgcac caaacacaaatcccctcaaa 28320 acaaaaattg gccatggcct agaatttgat tcaaacaagg ctatggttcctaaactagga 28380 actggcctta gttttgacag cacaggtgcc attacagtag gaaacaaaaataatgataag 28440 ctaactttgt ggaccacacc agctccatct cctaactgta gactaaatgcagagaaagat 28500 gctaaactca ctttggtctt aacaaaatgt ggcagtcaaa tacttgctacagtttcagtt 28560 ttggctgtta aaggcagttt ggctccaata tctggaacag ttcaaagtgctcatcttatt 28620 ataagatttg acgaaaatgg agtgctacta aacaattcct tcctggacccagaatattgg 28680 aactttagaa atggagatct tactgaaggc acagcctata caaacgctgttggatttatg 28740 cctaacctat cagcttatcc aaaatctcac ggtaaaactg ccaaaagtaacattgtcagt 28800 caagtttact taaacggaga caaaactaaa cctgtaacac taaccattacactaaacggt 28860 acacaggaaa caggagacac aactccaagt gcatactcta tgtcattttcatgggactgg 28920 tctggccaca actacattaa tgaaatattt gccacatcct cttacactttttcatacatt 28980 gcccaagaat aaagaatcgt ttgtgttatg tttcaacgtg tttatttttcaattgcagaa 29040 aatttcgaat catttttcat tcagtagtat agccccacca ccacatagcttatacagatc 29100 accgtacctt aatcaaactc acagaaccct agtattcaac ctgccacctccctcccaaca 29160 cacagagtac acagtccttt ctccccggct ggccttaaaa agcatcatatcatgggtaac 29220 agacatattc ttaggtgtta tattccacac ggtttcctgt cgagccaaacgctcatcagt 29280 gatattaata aactccccgg gcagctcact taagttcatg tcgctgtccagctgctgagc 29340 cacaggctgc tgtccaactt gcggttgctt aacgggcggc gaaggagaagtccacgccta 29400 catgggggta gagtcataat cgtgcatcag gatagggcgg tggtgctgcagcagcgcgcg 29460 aataaactgc tgccgccgcc gctccgtcct gcaggaatac aacatggcagtggtctcctc 29520 agcgatgatt cgcaccgccc gcagcataag gcgccttgtc ctccgggcacagcagcgcac 29580 cctgatctca cttaaatcag cacagtaact gcagcacagc accacaatattgttcaaaat 29640 cccacagtgc aaggcgctgt atccaaagct catggcgggg accacagaacccacgtggcc 29700 atcataccac aagcgcaggt agattaagtg gcgacccctc ataaacacgctggacataaa 29760 cattacctct tttggcatgt tgtaattcac cacctcccgg taccatataaacctctgatt 29820 aaacatggcg ccatccacca ccatcctaaa ccagctggcc aaaacctgcccgccggctat 29880 acactgcagg gaaccgggac tggaacaatg acagtggaga gcccaggactcgtaaccatg 29940 gatcatcatg ctcgtcatga tatcaatgtt ggcacaacac aggcacacgtgcatacactt 30000 cctcaggatt acaagctcct cccgcgttag aaccatatcc cagggaacaacccattcctg 30060 aatcagcgta aatcccacac tgcagggaag acctcgcacg taactcacgttgtgcattgt 30120 caaagtgtta cattcgggca gcagcggatg atcctccagt atggtagcgcgggtttctgt 30180 ctcaaaagga ggtagacgat ccctactgta cggagtgcgc cgagacaaccgagatcgtgt 30240 tggtcgtagt gtcatgccaa atggaacgcc ggacgtagtc atatttcctgaagcaaaacc 30300 aggtgcgggc gtgacaaaca gatctgcgtc tccggtctcg ccgcttagatcgctctgtgt 30360 agtagttgta gtatatccac tctctcaaag catccaggcg ccccctggcttcgggttcta 30420 tgtaaactcc ttcatgcgcc gctgccctga taacatccac caccgcagaataagccacac 30480 ccagccaacc tacacattcg ttctgcgagt cacacacggg aggagcgggaagagctggaa 30540 gaaccatgtt ttttttttta ttccaaaaga ttatccaaaa cctcaaaatgaagatctatt 30600 aagtgaacgc gctcccctcc ggtggcgtgg tcaaactcta cagccaaagaacagataatg 30660 gcatttgtaa gatgttgcac aatggcttcc aaaaggcaaa cggccctcacgtccaagtgg 30720 acgtaaaggc taaacccttc agggtgaatc tcctctataa acattccagcaccttcaacc 30780 atgcccaaat aattctcatc tcgccacctt ctcaatatat ctctaagcaaatcccgaata 30840 ttaagtccgg ccattgtaaa aatctgctcc agagcgccct ccaccttcagcctcaagcag 30900 cgaatcatga ttgcaaaaat tcaggttcct cacagacctg tataagattcaaaagcggaa 30960 cattaacaaa aataccgcga tcccgtaggt cccttcgcag ggccagctgaacataatcgt 31020 gcaggtctgc acggaccagc gcggccactt ccccgccagg aaccttgacaaaagaaccca 31080 cactgattat gacacgcata ctcggagcta tgctaaccag cgtagccccgatgtaagctt 31140 tgttgcatgg gcggcgatat aaaatgcaag gtgctgctca aaaaatcaggcaaagcctcg 31200 cgcaaaaaag aaagcacatc gtagtcatgc tcatgcagat aaaggcaggtaagctccgga 31260 accaccacag aaaaagacac catttttctc tcaaacatgt ctgcgggtttctgcataaac 31320 acaaaataaa ataacaaaaa aacatttaaa cattagaagc ctgtcttacaacaggaaaaa 31380 caacccttat aagcataaga cggactacgg ccatgccggc gtgaccgtaaaaaaactggt 31440 caccgtgatt aaaaagcacc accgacagct cctcggtcat gtccggagtcataatgtaag 31500 actcggtaaa cacatcaggt tgattcacat cggtcagtgc taaaaagcgaccgaaatagc 31560 ccgggggaat acatacccgc aggcgtagag acaacattac agcccccataggaggtataa 31620 caaaattaat aggagagaaa aacacataaa cacctgaaaa accctcctgcctaggcaaaa 31680 tagcaccctc ccgctccaga acaacataca gcgcttccac agcggcagccataacagtca 31740 gccttaccag taaaaaagaa aacctattaa aaaaacacca ctcgacacggcaccagctca 31800 atcagtcaca gtgtaaaaaa gggccaagtg cagagcgagt atatataggactaaaaaatg 31860 acgtaacggt taaagtccac aaaaaacacc cagaaaaccg cacgcgaacctacgcccaga 31920 aacgaaagcc aaaaaaccca caacttcctc aaatcgtcac ttccgttttcccacgttacg 31980 tcacttccca ttttaagaaa actacaattc ccaacacata caagttactccgccctaaaa 32040 cctacgtcac ccgccccgtt cccacgcccc gcgccacgtc acaaactccaccccctcatt 32100 atcatattgg cttcaatcca aaataaggta tattattgat gatgttaattaatttaaatc 32160 cgcatgcgat atcgagctct cccgggaatt cggatctgcg acgcgaggctggatggcctt 32220 ccccattatg attcttctcg cttccggcgg catcgggatg cccgcgttgcaggccatgct 32280 gtccaggcag gtagatgacg accatcaggg acagcttcac ggccagcaaaaggccaggaa 32340 ccgtaaaaag gccgcgttgc tggcgttttt ccataggctc cgcccccctgacgagcatca 32400 caaaaatcga cgctcaagtc agaggtggcg aaacccgaca ggactataaagataccaggc 32460 gtttccccct ggaagctccc tcgtgcgctc tcctgttccg accctgccgcttaccggata 32520 cctgtccgcc tttctccctt cgggaagcgt ggcgctttct caatgctcacgctgtaggta 32580 tctcagttcg gtgtaggtcg ttcgctccaa gctgggctgt gtgcacgaaccccccgttca 32640 gcccgaccgc tgcgccttat ccggtaacta tcgtcttgag tccaacccggtaagacacga 32700 cttatcgcca ctggcagcag ccactggtaa caggattagc agagcgaggtatgtaggcgg 32760 tgctacagag ttcttgaagt ggtggcctaa ctacggctac actagaaggacagtatttgg 32820 tatctgcgct ctgctgaagc cagttacctt cggaaaaaga gttggtagctcttgatccgg 32880 caaacaaacc accgctggta gcggtggttt ttttgtttgc aagcagcagattacgcgcag 32940 aaaaaaagga tctcaagaag atcctttgat cttttctacg gggtctgacgctcagtggaa 33000 cgaaaactca cgttaaggga ttttggtcat gagattatca aaaaggatcttcacctagat 33060 ccttttaaat caatctaaag tatatatgag taaacttggt ctgacagttaccaatgctta 33120 atcagtgagg cacctatctc agcgatctgt ctatttcgtt catccatagttgcctgactc 33180 cccgtcgtgt agataactac gatacgggag ggcttaccat ctggccccagtgctgcaatg 33240 ataccgcgag acccacgctc accggctcca gatttatcag caataaaccagccagccgga 33300 agggccgagc gcagaagtgg tcctgcaact ttatccgcct ccatccagtctattaattgt 33360 tgccgggaag ctagagtaag tagttcgcca gttaatagtt tgcgcaacgttgttgccatt 33420 gntgcaggca tcgtggtgtc acgctcgtcg tttggtatgg cttcattcagctccggttcc 33480 caacgatcaa ggcgagttac atgatccccc atgttgtgca aaaaagcggttagctccttc 33540 ggtcctccga tcgttgtcag aagtaagttg gccgcagtgt tatcactcatggttatggca 33600 gcactgcata attctcttac tgtcatgcca tccgtaagat gcttttctgtgactggtgag 33660 tactcaacca agtcattctg agaatagtgt atgcggcgac cgagttgctcttgcccggcg 33720 tcaacacggg ataataccgc gccacatagc agaactttaa aagtgctcatcattggaaaa 33780 cgttcttcgg ggcgaaaact ctcaaggatc ttaccgctgt tgagatccagttcgatgtaa 33840 cccactcgtg cacccaactg atcttcagca tcttttactt tcaccagcgtttctgggtga 33900 gcaaaaacag gaaggcaaaa tgccgcaaaa aagggaataa gggcgacacggaaatgttga 33960 atactcatac tcttcctttt tcaatattat tgaagcattt atcagggttattgtctcatg 34020 agcggataca tatttgaatg tatttagaaa aataaacaaa taggggttccgcgcacattt 34080 ccccgaaaag tgccacctga cgtctaagaa accattatta tcatgacattaacctataaa 34140 aataggcgta tcacgaggcc ctttcgtctt caaggatccg aattcccgggagagctcgat 34200 atcgcatgcg gatttaaatt aattaa 34226 86 954 DNA UnknownSau3A fragment used to construct vectors comprising suppressor tRNAsequences 86 ctagaggatc gaaaccatcc tctgctatat ggccgcatat attttacttgaagactagga 60 ccctacagaa aaggggtttt aaagtaggcg tgctaaacgt cagcggacctgacccgtgta 120 agaatccaca aggtatcctg gtggaaatgc gcatttgtag gcttcaatatctgtaatcct 180 actaattagg tgtggagagc tttcagccag tttcgtaggt ttggagaccatttaggggtt 240 ggcgtgtggc cccctcgtaa agtctttcgt acttcctaca tcagacaagtcttgcaattt 300 gcaatatctc ttttagccaa tatctaaatc tttaaaattt tgattttgttttttaaccag 360 gatgagagac attccagagt tgttaccttg tcaaaataaa caaatttaaagatgtctgtg 420 aaaagaaaca tatattcctc atgggaatat atccaggttg ttgaaggaggtacactcgag 480 tctccctatc agtgatagag atctcgaggt cgtagtcgtg gccgagtggttaaggcgatg 540 gactctaaat ccattggggt ctccccgcgc aggttcgaat cctgccgactacggcgtgct 600 ttttttactc tcgggtagag gaaatccggt gcactacctg tgcaatcacacagaataaca 660 tggagtagta ctttttattt tcctgttatt atctttctcc ataaaagtggaaccagataa 720 ttttagttct tttgtgtaac aagactagag attttttgaa gtgttacattggaaagcact 780 tgaaaacaca agtaatttct gacactgcta taaaaatgat ggaaaaacgctcaagttgtt 840 ttgcctttca gtcttcttga aatgctgtct ccctatctga aatccagctcacgtctgact 900 tccaaaaccg tgcttgcctt taacttatgg aataaatatc tcaaacagatcccc 954 87 34864 DNA Artificial Sequence pAd/PL-DEST 87 catcatcaataatatacctt attttggatt gaagccaata tgataatgag ggggtggagt 60 ttgtgacgtggcgcggggcg tgggaacggg gcgggtgacg tagtagtgtg gcggaagtgt 120 gatgttgcaagtgtggcgga acacatgtaa gcgacggatg tggcaaaagt gacgtttttg 180 gtgtgcgccggtgtacacag gaagtgacaa ttttcgcgcg gttttaggcg gatgttgtag 240 taaatttgggcgtaaccgag taagatttgg ccattttcgc gggaaaactg aataagagga 300 agtgaaatctgaataatttt gtgttactca tagcgcgtaa tatttgtcta gggccgcggg 360 gactttgaccgtttacgtgg agactcgccc aggtgttttt ctcaggtgtt ttccgcgttc 420 cgggtcaaagttggcgtttt attattatag tcagtcgaag cttggatccg gtacctctag 480 aattctcgagcggccgctag cgacatcgat cacaagtttg tacaaaaaag ctgaacgaga 540 aacgtaaaatgatataaata tcaatatatt aaattagatt ttgcataaaa aacagactac 600 ataatactgtaaaacacaac atatccagtc actatggcgg ccgcattagg caccccaggc 660 tttacactttatgcttccgg ctcgtataat gtgtggattt tgagttagga tccggcgaga 720 ttttcaggagctaaggaagc taaaatggag aaaaaaatca ctggatatac caccgttgat 780 atatcccaatggcatcgtaa agaacatttt gaggcatttc agtcagttgc tcaatgtacc 840 tataaccagaccgttcagct ggatattacg gcctttttaa agaccgtaaa gaaaaataag 900 cacaagttttatccggcctt tattcacatt cttgcccgcc tgatgaatgc tcatccggaa 960 ttccgtatggcaatgaaaga cggtgagctg gtgatatggg atagtgttca cccttgttac 1020 accgttttccatgagcaaac tgaaacgttt tcatcgctct ggagtgaata ccacgacgat 1080 ttccggcagtttctacacat atattcgcaa gatgtggcgt gttacggtga aaacctggcc 1140 tatttccctaaagggtttat tgagaatatg tttttcgtct cagccaatcc ctgggtgagt 1200 ttcaccagttttgatttaaa cgtggccaat atggacaact tcttcgcccc cgttttcacc 1260 atgggcaaatattatacgca aggcgacaag gtgctgatgc cgctggcgat tcaggttcat 1320 catgccgtctgtgatggctt ccatgtcggc agaatgctta atgaattaca acagtactgc 1380 gatgagtggcagggcggggc gtaaacgcgt ggatccggct tactaaaagc cagataacag 1440 tatgcgtatttgcgcgctga tttttgcggt ataagaatat atactgatat gtatacccga 1500 agtatgtcaaaaagaggtgt gctatgaagc agcgtattac agtgacagtt gacagcgaca 1560 gctatcagttgctcaaggca tatatgatgt caatatctcc ggtctggtaa gcacaaccat 1620 gcagaatgaagcccgtcgtc tgcgtgccga acgctggaaa gcggaaaatc aggaagggat 1680 ggctgaggtcgcccggttta ttgaaatgaa cggctctttt gctgacgaga acagggactg 1740 gtgaaatgcagtttaaggtt tacacctata aaagagagag ccgttatcgt ctgtttgtgg 1800 atgtacagagtgatattatt gacacgcccg ggcgacggat ggtgatcccc ctggccagtg 1860 cacgtctgctgtcagataaa gtctcccgtg aactttaccc ggtggtgcat atcggggatg 1920 aaagctggcgcatgatgacc accgatatgg ccagtgtgcc ggtctccgtt atcggggaag 1980 aagtggctgatctcagccac cgcgaaaatg acatcaaaaa cgccattaac ctgatgttct 2040 ggggaatataaatgtcaggc tccgttatac acagccagtc tgcaggtcga ccatagtgac 2100 tggatatgttgtgttttaca gtattatgta gtctgttttt tatgcaaaat ctaatttaat 2160 atattgatatttatatcatt ttacgtttct cgttcagctt tcttgtacaa agtggtgatc 2220 gattcgacagatcactgaaa tgtgtgggcg tggcttaagg gtgggaaaga atatataagg 2280 tgggggtcttatgtagtttt gtatctgttt tgcagcagcc gccgccgcca tgagcaccaa 2340 ctcgtttgatggaagcattg tgagctcata tttgacaacg cgcatgcccc catgggccgg 2400 ggtgcgtcagaatgtgatgg gctccagcat tgatggtcgc cccgtcctgc ccgcaaactc 2460 tactaccttgacctacgaga ccgtgtctgg aacgccgttg gagactgcag cctccgccgc 2520 cgcttcagccgctgcagcca ccgcccgcgg gattgtgact gactttgctt tcctgagccc 2580 gcttgcaagcagtgcagctt cccgttcatc cgcccgcgat gacaagttga cggctctttt 2640 ggcacaattggattctttga cccgggaact taatgtcgtt tctcagcagc tgttggatct 2700 gcgccagcaggtttctgccc tgaaggcttc ctcccctccc aatgcggttt aaaacataaa 2760 taaaaaaccagactctgttt ggatttggat caagcaagtg tcttgctgtc tttatttagg 2820 ggttttgcgcgcgcggtagg cccgggacca gcggtctcgg tcgttgaggg tcctgtgtat 2880 tttttccaggacgtggtaaa ggtgactctg gatgttcaga tacatgggca taagcccgtc 2940 tctggggtggaggtagcacc actgcagagc ttcatgctgc ggggtggtgt tgtagatgat 3000 ccagtcgtagcaggagcgct gggcgtggtg cctaaaaatg tctttcagta gcaagctgat 3060 tgccaggggcaggcccttgg tgtaagtgtt tacaaagcgg ttaagctggg atgggtgcat 3120 acgtggggatatgagatgca tcttggactg tatttttagg ttggctatgt tcccagccat 3180 atccctccggggattcatgt tgtgcagaac caccagcaca gtgtatccgg tgcacttggg 3240 aaatttgtcatgtagcttag aaggaaatgc gtggaagaac ttggagacgc ccttgtgacc 3300 tccaagattttccatgcatt cgtccataat gatggcaatg ggcccacggg cggcggcctg 3360 ggcgaagatatttctgggat cactaacgtc atagttgtgt tccaggatga gatcgtcata 3420 ggccatttttacaaagcgcg ggcggagggt gccagactgc ggtataatgg ttccatccgg 3480 cccaggggcgtagttaccct cacagatttg catttcccac gctttgagtt cagatggggg 3540 gatcatgtctacctgcgggg cgatgaagaa aacggtttcc ggggtagggg agatcagctg 3600 ggaagaaagcaggttcctga gcagctgcga cttaccgcag ccggtgggcc cgtaaatcac 3660 acctattaccgggtgcaact ggtagttaag agagctgcag ctgccgtcat ccctgagcag 3720 gggggccacttcgttaagca tgtccctgac tcgcatgttt tccctgacca aatccgccag 3780 aaggcgctcgccgcccagcg atagcagttc ttgcaaggaa gcaaagtttt tcaacggttt 3840 gagaccgtccgccgtaggca tgcttttgag cgtttgacca agcagttcca ggcggtccca 3900 cagctcggtcacctgctcta cggcatctcg atccagcata tctcctcgtt tcgcgggttg 3960 gggcggctttcgctgtacgg cagtagtcgg tgctcgtcca gacgggccag ggtcatgtct 4020 ttccacgggcgcagggtcct cgtcagcgta gtctgggtca cggtgaaggg gtgcgctccg 4080 ggctgcgcgctggccagggt gcgcttgagg ctggtcctgc tggtgctgaa gcgctgccgg 4140 tcttcgccctgcgcgtcggc caggtagcat ttgaccatgg tgtcatagtc cagcccctcc 4200 gcggcgtggcccttggcgcg cagcttgccc ttggaggagg cgccgcacga ggggcagtgc 4260 agacttttgagggcgtagag cttgggcgcg agaaataccg attccgggga gtaggcatcc 4320 gcgccgcaggccccgcagac ggtctcgcat tccacgagcc aggtgagctc tggccgttcg 4380 gggtcaaaaaccaggtttcc cccatgcttt ttgatgcgtt tcttacctct ggtttccatg 4440 agccggtgtccacgctcggt gacgaaaagg ctgtccgtgt ccccgtatac agacttgaga 4500 ggcctgtcctcgagcggtgt tccgcggtcc tcctcgtata gaaactcgga ccactctgag 4560 acaaaggctcgcgtccaggc cagcacgaag gaggctaagt gggaggggta gcggtcgttg 4620 tccactagggggtccactcg ctccagggtg tgaagacaca tgtcgccctc ttcggcatca 4680 aggaaggtgattggtttgta ggtgtaggcc acgtgaccgg gtgttcctga aggggggcta 4740 taaaagggggtgggggcgcg ttcgtcctca ctctcttccg catcgctgtc tgcgagggcc 4800 agctgttggggtgagtactc cctctgaaaa gcgggcatga cttctgcgct aagattgtca 4860 gtttccaaaaacgaggagga tttgatattc acctggcccg cggtgatgcc tttgagggtg 4920 gccgcatccatctggtcaga aaagacaatc tttttgttgt caagcttggt ggcaaacgac 4980 ccgtagagggcgttggacag caacttggcg atggagcgca gggtttggtt tttgtcgcga 5040 tcggcgcgctccttggccgc gatgtttagc tgcacgtatt cgcgcgcaac gcaccgccat 5100 tcgggaaagacggtggtgcg ctcgtcgggc accaggtgca cgcgccaacc gcggttgtgc 5160 agggtgacaaggtcaacgct ggtggctacc tctccgcgta ggcgctcgtt ggtccagcag 5220 aggcggccgcccttgcgcga gcagaatggc ggtagggggt ctagctgcgt ctcgtccggg 5280 gggtctgcgtccacggtaaa gaccccgggc agcaggcgcg cgtcgaagta gtctatcttg 5340 catccttgcaagtctagcgc ctgctgccat gcgcgggcgg caagcgcgcg ctcgtatggg 5400 ttgagtgggggaccccatgg catggggtgg gtgagcgcgg aggcgtacat gccgcaaatg 5460 tcgtaaacgtagaggggctc tctgagtatt ccaagatatg tagggtagca tcttccaccg 5520 cggatgctggcgcgcacgta atcgtatagt tcgtgcgagg gagcgaggag gtcgggaccg 5580 aggttgctacgggcgggctg ctctgctcgg aagactatct gcctgaagat ggcatgtgag 5640 ttggatgatatggttggacg ctggaagacg ttgaagctgg cgtctgtgag acctaccgcg 5700 tcacgcacgaaggaggcgta ggagtcgcgc agcttgttga ccagctcggc ggtgacctgc 5760 acgtctagggcgcagtagtc cagggtttcc ttgatgatgt catacttatc ctgtcccttt 5820 tttttccacagctcgcggtt gaggacaaac tcttcgcggt ctttccagta ctcttggatc 5880 ggaaacccgtcggcctccga acggtaagag cctagcatgt agaactggtt gacggcctgg 5940 taggcgcagcatcccttttc tacgggtagc gcgtatgcct gcgcggcctt ccggagcgag 6000 gtgtgggtgagcgcaaaggt gtccctgacc atgactttga ggtactggta tttgaagtca 6060 gtgtcgtcgcatccgccctg ctcccagagc aaaaagtccg tgcgcttttt ggaacgcgga 6120 tttggcagggcgaaggtgac atcgttgaag agtatctttc ccgcgcgagg cataaagttg 6180 cgtgtgatgcggaagggtcc cggcacctcg gaacggttgt taattacctg ggcggcgagc 6240 acgatctcgtcaaagccgtt gatgttgtgg cccacaatgt aaagttccaa gaagcgcggg 6300 atgcccttgatggaaggcaa ttttttaagt tcctcgtagg tgagctcttc aggggagctg 6360 agcccgtgctctgaaagggc ccagtctgca agatgagggt tggaagcgac gaatgagctc 6420 cacaggtcacgggccattag catttgcagg tggtcgcgaa aggtcctaaa ctggcgacct 6480 atggccattttttctggggt gatgcagtag aaggtaagcg ggtcttgttc ccagcggtcc 6540 catccaaggttcgcggctag gtctcgcgcg gcagtcacta gaggctcatc tccgccgaac 6600 ttcatgaccagcatgaaggg cacgagctgc ttcccaaagg cccccatcca agtataggtc 6660 tctacatcgtaggtgacaaa gagacgctcg gtgcgaggat gcgagccgat cgggaagaac 6720 tggatctcccgccaccaatt ggaggagtgg ctattgatgt ggtgaaagta gaagtccctg 6780 cgacgggccgaacactcgtg ctggcttttg taaaaacgtg cgcagtactg gcagcggtgc 6840 acgggctgtacatcctgcac gaggttgacc tgacgaccgc gcacaaggaa gcagagtggg 6900 aatttgagcccctcgcctgg cgggtttggc tggtggtctt ctacttcggc tgcttgtcct 6960 tgaccgtctggctgctcgag gggagttacg gtggatcgga ccaccacgcc gcgcgagccc 7020 aaagtccagatgtccgcgcg cggcggtcgg agcttgatga caacatcgcg cagatgggag 7080 ctgtccatggtctggagctc ccgcggcgtc aggtcaggcg ggagctcctg caggtttacc 7140 tcgcatagacgggtcagggc gcgggctaga tccaggtgat acctaatttc caggggctgg 7200 ttggtggcggcgtcgatggc ttgcaagagg ccgcatcccc gcggcgcgac tacggtaccg 7260 cgcggcgggcggtgggccgc gggggtgtcc ttggatgatg catctaaaag cggtgacgcg 7320 ggcgagcccccggaggtagg gggggctccg gacccgccgg gagagggggc aggggcacgt 7380 cggcgccgcgcgcgggcagg agctggtgct gcgcgcgtag gttgctggcg aacgcgacga 7440 cgcggcggttgatctcctga atctggcgcc tctgcgtgaa gacgacgggc ccggtgagct 7500 tgagcctgaaagagagttcg acagaatcaa tttcggtgtc gttgacggcg gcctggcgca 7560 aaatctcctgcacgtctcct gagttgtctt gataggcgat ctcggccatg aactgctcga 7620 tctcttcctcctggagatct ccgcgtccgg ctcgctccac ggtggcggcg aggtcgttgg 7680 aaatgcgggccatgagctgc gagaaggcgt tgaggcctcc ctcgttccag acgcggctgt 7740 agaccacgcccccttcggca tcgcgggcgc gcatgaccac ctgcgcgaga ttgagctcca 7800 cgtgccgggcgaagacggcg tagtttcgca ggcgctgaaa gaggtagttg agggtggtgg 7860 cggtgtgttctgccacgaag aagtacataa cccagcgtcg caacgtggat tcgttgatat 7920 cccccaaggcctcaaggcgc tccatggcct cgtagaagtc cacggcgaag ttgaaaaact 7980 gggagttgcgcgccgacacg gttaactcct cctccagaag acggatgagc tcggcgacag 8040 tgtcgcgcacctcgcgctca aaggctacag gggcctcttc ttcttcttca atctcctctt 8100 ccataagggcctccccttct tcttcttctg gcggcggtgg gggagggggg acacggcggc 8160 gacgacggcgcaccgggagg cggtcgacaa agcgctcgat catctccccg cggcgacggc 8220 gcatggtctcggtgacggcg cggccgttct cgcgggggcg cagttggaag acgccgcccg 8280 tcatgtcccggttatgggtt ggcggggggc tgccatgcgg cagggatacg gcgctaacga 8340 tgcatctcaacaattgttgt gtaggtactc cgccgccgag ggacctgagc gagtccgcat 8400 cgaccggatcggaaaacctc tcgagaaagg cgtctaacca gtcacagtcg caaggtaggc 8460 tgagcaccgtggcgggcggc agcgggcggc ggtcggggtt gtttctggcg gaggtgctgc 8520 tgatgatgtaattaaagtag gcggtcttga gacggcggat ggtcgacaga agcaccatgt 8580 ccttgggtccggcctgctga atgcgcaggc ggtcggccat gccccaggct tcgttttgac 8640 atcggcgcaggtctttgtag tagtcttgca tgagcctttc taccggcact tcttcttctc 8700 cttcctcttgtcctgcatct cttgcatcta tcgctgcggc ggcggcggag tttggccgta 8760 ggtggcgccctcttcctccc atgcgtgtga ccccgaagcc cctcatcggc tgaagcaggg 8820 ctaggtcggcgacaacgcgc tcggctaata tggcctgctg cacctgcgtg agggtagact 8880 ggaagtcatccatgtccaca aagcggtggt atgcgcccgt gttgatggtg taagtgcagt 8940 tggccataacggaccagtta acggtctggt gacccggctg cgagagctcg gtgtacctga 9000 gacgcgagtaagccctcgag tcaaatacgt agtcgttgca agtccgcacc aggtactggt 9060 atcccaccaaaaagtgcggc ggcggctggc ggtagagggg ccagcgtagg gtggccgggg 9120 ctccgggggcgagatcttcc aacataaggc gatgatatcc gtagatgtac ctggacatcc 9180 aggtgatgccggcggcggtg gtggaggcgc gcggaaagtc gcggacgcgg ttccagatgt 9240 tgcgcagcggcaaaaagtgc tccatggtcg ggacgctctg gccggtcagg cgcgcgcaat 9300 cgttgacgctctagaccgtg caaaaggaga gcctgtaagc gggcactctt ccgtggtctg 9360 gtggataaattcgcaagggt atcatggcgg acgaccgggg ttcgagcccc gtatccggcc 9420 gtccgccgtgatccatgcgg ttaccgcccg cgtgtcgaac ccaggtgtgc gacgtcagac 9480 aacgggggagtgctcctttt ggcttccttc caggcgcggc ggctgctgcg ctagcttttt 9540 tggccactggccgcgcgcag cgtaagcggt taggctggaa agcgaaagca ttaagtggct 9600 cgctccctgtagccggaggg ttattttcca agggttgagt cgcgggaccc ccggttcgag 9660 tctcggaccggccggactgc ggcgaacggg ggtttgcctc cccgtcatgc aagaccccgc 9720 ttgcaaattcctccggaaac agggacgagc cccttttttg cttttcccag atgcatccgg 9780 tgctgcggcagatgcgcccc cctcctcagc agcggcaaga gcaagagcag cggcagacat 9840 gcagggcaccctcccctcct cctaccgcgt caggaggggc gacatccgcg gttgacgcgg 9900 cagcagatggtgattacgaa cccccgcggc gccgggcccg gcactacctg gacttggagg 9960 agggcgagggcctggcgcgg ctaggagcgc cctctcctga gcggtaccca agggtgcagc 10020 tgaagcgtgatacgcgtgag gcgtacgtgc cgcggcagaa cctgtttcgc gaccgcgagg 10080 gagaggagcccgaggagatg cgggatcgaa agttccacgc agggcgcgag ctgcggcatg 10140 gcctgaatcgcgagcggttg ctgcgcgagg aggactttga gcccgacgcg cgaaccggga 10200 ttagtcccgcgcgcgcacac gtggcggccg ccgacctggt aaccgcatac gagcagacgg 10260 tgaaccaggagattaacttt caaaaaagct ttaacaacca cgtgcgtacg cttgtggcgc 10320 gcgaggaggtggctatagga ctgatgcatc tgtgggactt tgtaagcgcg ctggagcaaa 10380 acccaaatagcaagccgctc atggcgcagc tgttccttat agtgcagcac agcagggaca 10440 acgaggcattcagggatgcg ctgctaaaca tagtagagcc cgagggccgc tggctgctcg 10500 atttgataaacatcctgcag agcatagtgg tgcaggagcg cagcttgagc ctggctgaca 10560 aggtggccgccatcaactat tccatgctta gcctgggcaa gttttacgcc cgcaagatat 10620 accataccccttacgttccc atagacaagg aggtaaagat cgaggggttc tacatgcgca 10680 tggcgctgaaggtgcttacc ttgagcgacg acctgggcgt ttatcgcaac gagcgcatcc 10740 acaaggccgtgagcgtgagc cggcggcgcg agctcagcga ccgcgagctg atgcacagcc 10800 tgcaaagggccctggctggc acgggcagcg gcgatagaga ggccgagtcc tactttgacg 10860 cgggcgctgacctgcgctgg gccccaagcc gacgcgccct ggaggcagct ggggccggac 10920 ctgggctggcggtggcaccc gcgcgcgctg gcaacgtcgg cggcgtggag gaatatgacg 10980 aggacgatgagtacgagcca gaggacggcg agtactaagc ggtgatgttt ctgatcagat 11040 gatgcaagacgcaacggacc cggcggtgcg ggcggcgctg cagagccagc cgtccggcct 11100 taactccacggacgactggc gccaggtcat ggaccgcatc atgtcgctga ctgcgcgcaa 11160 tcctgacgcgttccggcagc agccgcaggc caaccggctc tccgcaattc tggaagcggt 11220 ggtcccggcgcgcgcaaacc ccacgcacga gaaggtgctg gcgatcgtaa acgcgctggc 11280 cgaaaacagggccatccggc ccgacgaggc cggcctggtc tacgacgcgc tgcttcagcg 11340 cgtggctcgttacaacagcg gcaacgtgca gaccaacctg gaccggctgg tgggggatgt 11400 gcgcgaggccgtggcgcagc gtgagcgcgc gcagcagcag ggcaacctgg gctccatggt 11460 tgcactaaacgccttcctga gtacacagcc cgccaacgtg ccgcggggac aggaggacta 11520 caccaactttgtgagcgcac tgcggctaat ggtgactgag acaccgcaaa gtgaggtgta 11580 ccagtctgggccagactatt ttttccagac cagtagacaa ggcctgcaga ccgtaaacct 11640 gagccaggctttcaaaaact tgcaggggct gtggggggtg cgggctccca caggcgaccg 11700 cgcgaccgtgtctagcttgc tgacgcccaa ctcgcgcctg ttgctgctgc taatagcgcc 11760 cttcacggacagtggcagcg tgtcccggga cacataccta ggtcacttgc tgacactgta 11820 ccgcgaggccataggtcagg cgcatgtgga cgagcatact ttccaggaga ttacaagtgt 11880 cagccgcgcgctggggcagg aggacacggg cagcctggag gcaaccctaa actacctgct 11940 gaccaaccggcggcagaaga tcccctcgtt gcacagttta aacagcgagg aggagcgcat 12000 tttgcgctacgtgcagcaga gcgtgagcct taacctgatg cgcgacgggg taacgcccag 12060 cgtggcgctggacatgaccg cgcgcaacat ggaaccgggc atgtatgcct caaaccggcc 12120 gtttatcaaccgcctaatgg actacttgca tcgcgcggcc gccgtgaacc ccgagtattt 12180 caccaatgccatcttgaacc cgcactggct accgccccct ggtttctaca ccgggggatt 12240 cgaggtgcccgagggtaacg atggattcct ctgggacgac atagacgaca gcgtgttttc 12300 cccgcaaccgcagaccctgc tagagttgca acagcgcgag caggcagagg cggcgctgcg 12360 aaaggaaagcttccgcaggc caagcagctt gtccgatcta ggcgctgcgg ccccgcggtc 12420 agatgctagtagcccatttc caagcttgat agggtctctt accagcactc gcaccacccg 12480 cccgcgcctgctgggcgagg aggagtacct aaacaactcg ctgctgcagc cgcagcgcga 12540 aaaaaacctgcctccggcat ttcccaacaa cgggatagag agcctagtgg acaagatgag 12600 tagatggaagacgtacgcgc aggagcacag ggacgtgcca ggcccgcgcc cgcccacccg 12660 tcgtcaaaggcacgaccgtc agcggggtct ggtgtgggag gacgatgact cggcagacga 12720 cagcagcgtcctggatttgg gagggagtgg caacccgttt gcgcaccttc gccccaggct 12780 ggggagaatgttttaaaaaa aaaaaagcat gatgcaaaat aaaaaactca ccaaggccat 12840 ggcaccgagcgttggttttc ttgtattccc cttagtatgc ggcgcgcggc gatgtatgag 12900 gaaggtcctcctccctccta cgagagtgtg gtgagcgcgg cgccagtggc ggcggcgctg 12960 ggttctcccttcgatgctcc cctggacccg ccgtttgtgc ctccgcggta cctgcggcct 13020 accggggggagaaacagcat ccgttactct gagttggcac ccctattcga caccacccgt 13080 gtgtacctggtggacaacaa gtcaacggat gtggcatccc tgaactacca gaacgaccac 13140 agcaactttctgaccacggt cattcaaaac aatgactaca gcccggggga ggcaagcaca 13200 cagaccatcaatcttgacga ccggtcgcac tggggcggcg acctgaaaac catcctgcat 13260 accaacatgccaaatgtgaa cgagttcatg tttaccaata agtttaaggc gcgggtgatg 13320 gtgtcgcgcttgcctactaa ggacaatcag gtggagctga aatacgagtg ggtggagttc 13380 acgctgcccgagggcaacta ctccgagacc atgaccatag accttatgaa caacgcgatc 13440 gtggagcactacttgaaagt gggcagacag aacggggttc tggaaagcga catcggggta 13500 aagtttgacacccgcaactt cagactgggg tttgaccccg tcactggtct tgtcatgcct 13560 ggggtatatacaaacgaagc cttccatcca gacatcattt tgctgccagg atgcggggtg 13620 gacttcacccacagccgcct gagcaacttg ttgggcatcc gcaagcggca acccttccag 13680 gagggctttaggatcaccta cgatgatctg gagggtggta acattcccgc actgttggat 13740 gtggacgcctaccaggcgag cttgaaagat gacaccgaac agggcggggg tggcgcaggc 13800 ggcagcaacagcagtggcag cggcgcggaa gagaactcca acgcggcagc cgcggcaatg 13860 cagccggtggaggacatgaa cgatcatgcc attcgcggcg acacctttgc cacacgggct 13920 gaggagaagcgcgctgaggc cgaagcagcg gccgaagctg ccgcccccgc tgcgcaaccc 13980 gaggtcgagaagcctcagaa gaaaccggtg atcaaacccc tgacagagga cagcaagaaa 14040 cgcagttacaacctaataag caatgacagc accttcaccc agtaccgcag ctggtacctt 14100 gcatacaactacggcgaccc tcagaccgga atccgctcat ggaccctgct ttgcactcct 14160 gacgtaacctgcggctcgga gcaggtctac tggtcgttgc cagacatgat gcaagacccc 14220 gtgaccttccgctccacgcg ccagatcagc aactttccgg tggtgggcgc cgagctgttg 14280 cccgtgcactccaagagctt ctacaacgac caggccgtct actcccaact catccgccag 14340 tttacctctctgacccacgt gttcaatcgc tttcccgaga accagatttt ggcgcgcccg 14400 ccagcccccaccatcaccac cgtcagtgaa aacgttcctg ctctcacaga tcacgggacg 14460 ctaccgctgcgcaacagcat cggaggagtc cagcgagtga ccattactga cgccagacgc 14520 cgcacctgcccctacgttta caaggccctg ggcatagtct cgccgcgcgt cctatcgagc 14580 cgcactttttgagcaagcat gtccatcctt atatcgccca gcaataacac aggctggggc 14640 ctgcgcttcccaagcaagat gtttggcggg gccaagaagc gctccgacca acacccagtg 14700 cgcgtgcgcgggcactaccg cgcgccctgg ggcgcgcaca aacgcggccg cactgggcgc 14760 accaccgtcgatgacgccat cgacgcggtg gtggaggagg cgcgcaacta cacgcccacg 14820 ccgccaccagtgtccacagt ggacgcggcc attcagaccg tggtgcgcgg agcccggcgc 14880 tatgctaaaatgaagagacg gcggaggcgc gtagcacgtc gccaccgccg ccgacccggc 14940 actgccgcccaacgcgcggc ggcggccctg cttaaccgcg cacgtcgcac cggccgacgg 15000 gcggccatgcgggccgctcg aaggctggcc gcgggtattg tcactgtgcc ccccaggtcc 15060 aggcgacgagcggccgccgc agcagccgcg gccattagtg ctatgactca gggtcgcagg 15120 ggcaacgtgtattgggtgcg cgactcggtt agcggcctgc gcgtgcccgt gcgcacccgc 15180 cccccgcgcaactagattgc aagaaaaaac tacttagact cgtactgttg tatgtatcca 15240 gcggcggcggcgcgcaacga agctatgtcc aagcgcaaaa tcaaagaaga gatgctccag 15300 gtcatcgcgccggagatcta tggccccccg aagaaggaag agcaggatta caagccccga 15360 aagctaaagcgggtcaaaaa gaaaaagaaa gatgatgatg atgaacttga cgacgaggtg 15420 gaactgctgcacgctaccgc gcccaggcga cgggtacagt ggaaaggtcg acgcgtaaaa 15480 cgtgttttgcgacccggcac caccgtagtc tttacgcccg gtgagcgctc cacccgcacc 15540 tacaagcgcgtgtatgatga ggtgtacggc gacgaggacc tgcttgagca ggccaacgag 15600 cgcctcggggagtttgccta cggaaagcgg cataaggaca tgctggcgtt gccgctggac 15660 gagggcaacccaacacctag cctaaagccc gtaacactgc agcaggtgct gcccgcgctt 15720 gcaccgtccgaagaaaagcg cggcctaaag cgcgagtctg gtgacttggc acccaccgtg 15780 cagctgatggtacccaagcg ccagcgactg gaagatgtct tggaaaaaat gaccgtggaa 15840 cctgggctggagcccgaggt ccgcgtgcgg ccaatcaagc aggtggcgcc gggactgggc 15900 gtgcagaccgtggacgttca gatacccact accagtagca ccagtattgc caccgccaca 15960 gagggcatggagacacaaac gtccccggtt gcctcagcgg tggcggatgc cgcggtgcag 16020 gcggtcgctgcggccgcgtc caagacctct acggaggtgc aaacggaccc gtggatgttt 16080 cgcgtttcagccccccggcg cccgcgcggt tcgaggaagt acggcgccgc cagcgcgcta 16140 ctgcccgaatatgccctaca tccttccatt gcgcctaccc ccggctatcg tggctacacc 16200 taccgccccagaagacgagc aactacccga cgccgaacca ccactggaac ccgccgccgc 16260 cgtcgccgtcgccagcccgt gctggccccg atttccgtgc gcagggtggc tcgcgaagga 16320 ggcaggaccctggtgctgcc aacagcgcgc taccacccca gcatcgttta aaagccggtc 16380 tttgtggttcttgcagatat ggccctcacc tgccgcctcc gtttcccggt gccgggattc 16440 cgaggaagaatgcaccgtag gaggggcatg gccggccacg gcctgacggg cggcatgcgt 16500 cgtgcgcaccaccggcggcg gcgcgcgtcg caccgtcgca tgcgcggcgg tatcctgccc 16560 ctccttattccactgatcgc cgcggcgatt ggcgccgtgc ccggaattgc atccgtggcc 16620 ttgcaggcgcagagacactg attaaaaaca agttgcatgt ggaaaaatca aaataaaaag 16680 tctggactctcacgctcgct tggtcctgta actattttgt agaatggaag acatcaactt 16740 tgcgtctctggccccgcgac acggctcgcg cccgttcatg ggaaactggc aagatatcgg 16800 caccagcaatatgagcggtg gcgccttcag ctggggctcg ctgtggagcg gcattaaaaa 16860 tttcggttccaccgttaaga actatggcag caaggcctgg aacagcagca caggccagat 16920 gctgagggataagttgaaag agcaaaattt ccaacaaaag gtggtagatg gcctggcctc 16980 tggcattagcggggtggtgg acctggccaa ccaggcagtg caaaataaga ttaacagtaa 17040 gcttgatccccgccctcccg tagaggagcc tccaccggcc gtggagacag tgtctccaga 17100 ggggcgtggcgaaaagcgtc cgcgccccga cagggaagaa actctggtga cgcaaataga 17160 cgagcctccctcgtacgagg aggcactaaa gcaaggcctg cccaccaccc gtcccatcgc 17220 gcccatggctaccggagtgc tgggccagca cacacccgta acgctggacc tgcctccccc 17280 cgccgacacccagcagaaac ctgtgctgcc aggcccgacc gccgttgttg taacccgtcc 17340 tagccgcgcgtccctgcgcc gcgccgccag cggtccgcga tcgttgcggc ccgtagccag 17400 tggcaactggcaaagcacac tgaacagcat cgtgggtctg ggggtgcaat ccctgaagcg 17460 ccgacgatgcttctgaatag ctaacgtgtc gtatgtgtgt catgtatgcg tccatgtcgc 17520 cgccagaggagctgctgagc cgccgcgcgc ccgctttcca agatggctac cccttcgatg 17580 atgccgcagtggtcttacat gcacatctcg ggccaggacg cctcggagta cctgagcccc 17640 gggctggtgcagtttgcccg cgccaccgag acgtacttca gcctgaataa caagtttaga 17700 aaccccacggtggcgcctac gcacgacgtg accacagacc ggtcccagcg tttgacgctg 17760 cggttcatccctgtggaccg tgaggatact gcgtactcgt acaaggcgcg gttcacccta 17820 gctgtgggtgataaccgtgt gctggacatg gcttccacgt actttgacat ccgcggcgtg 17880 ctggacaggggccctacttt taagccctac tctggcactg cctacaacgc cctggctccc 17940 aagggtgccccaaatccttg cgaatgggat gaagctgcta ctgctcttga aataaaccta 18000 gaagaagaggacgatgacaa cgaagacgaa gtagacgagc aagctgagca gcaaaaaact 18060 cacgtatttgggcaggcgcc ttattctggt ataaatatta caaaggaggg tattcaaata 18120 ggtgtcgaaggtcaaacacc taaatatgcc gataaaacat ttcaacctga acctcaaata 18180 ggagaatctcagtggtacga aactgaaatt aatcatgcag ctgggagagt ccttaaaaag 18240 actaccccaatgaaaccatg ttacggttca tatgcaaaac ccacaaatga aaatggaggg 18300 caaggcattcttgtaaagca acaaaatgga aagctagaaa gtcaagtgga aatgcaattt 18360 ttctcaactactgaggcgac cgcaggcaat ggtgataact tgactcctaa agtggtattg 18420 tacagtgaagatgtagatat agaaacccca gacactcata tttcttacat gcccactatt 18480 aaggaaggtaactcacgaga actaatgggc caacaatcta tgcccaacag gcctaattac 18540 attgcttttagggacaattt tattggtcta atgtattaca acagcacggg taatatgggt 18600 gttctggcgggccaagcatc gcagttgaat gctgttgtag atttgcaaga cagaaacaca 18660 gagctttcataccagctttt gcttgattcc attggtgata gaaccaggta cttttctatg 18720 tggaatcaggctgttgacag ctatgatcca gatgttagaa ttattgaaaa tcatggaact 18780 gaagatgaacttccaaatta ctgctttcca ctgggaggtg tgattaatac agagactctt 18840 accaaggtaaaacctaaaac aggtcaggaa aatggatggg aaaaagatgc tacagaattt 18900 tcagataaaaatgaaataag agttggaaat aattttgcca tggaaatcaa tctaaatgcc 18960 aacctgtggagaaatttcct gtactccaac atagcgctgt atttgcccga caagctaaag 19020 tacagtccttccaacgtaaa aatttctgat aacccaaaca cctacgacta catgaacaag 19080 cgagtggtggctcccgggtt agtggactgc tacattaacc ttggagcacg ctggtccctt 19140 gactatatggacaacgtcaa cccatttaac caccaccgca atgctggcct gcgctaccgc 19200 tcaatgttgctgggcaatgg tcgctatgtg cccttccaca tccaggtgcc tcagaagttc 19260 tttgccattaaaaacctcct tctcctgccg ggctcataca cctacgagtg gaacttcagg 19320 aaggatgttaacatggttct gcagagctcc ctaggaaatg acctaagggt tgacggagcc 19380 agcattaagtttgatagcat ttgcctttac gccaccttct tccccatggc ccacaacacc 19440 gcctccacgcttgaggccat gcttagaaac gacaccaacg accagtcctt taacgactat 19500 ctctccgccgccaacatgct ctaccctata cccgccaacg ctaccaacgt gcccatatcc 19560 atcccctcccgcaactgggc ggctttccgc ggctgggcct tcacgcgcct taagactaag 19620 gaaaccccatcactgggctc gggctacgac ccttattaca cctactctgg ctctataccc 19680 tacctagatggaacctttta cctcaaccac acctttaaga aggtggccat tacctttgac 19740 tcttctgtcagctggcctgg caatgaccgc ctgcttaccc ccaacgagtt tgaaattaag 19800 cgctcagttgacggggaggg ttacaacgtt gcccagtgta acatgaccaa agactggttc 19860 ctggtacaaatgctagctaa ctacaacatt ggctaccagg gcttctatat cccagagagc 19920 tacaaggaccgcatgtactc cttctttaga aacttccagc ccatgagccg tcaggtggtg 19980 gatgatactaaatacaagga ctaccaacag gtgggcatcc tacaccaaca caacaactct 20040 ggatttgttggctaccttgc ccccaccatg cgcgaaggac aggcctaccc tgctaacttc 20100 ccctatccgcttataggcaa gaccgcagtt gacagcatta cccagaaaaa gtttctttgc 20160 gatcgcaccctttggcgcat cccattctcc agtaacttta tgtccatggg cgcactcaca 20220 gacctgggccaaaaccttct ctacgccaac tccgcccacg cgctagacat gacttttgag 20280 gtggatcccatggacgagcc cacccttctt tatgttttgt ttgaagtctt tgacgtggtc 20340 cgtgtgcaccggccgcaccg cggcgtcatc gaaaccgtgt acctgcgcac gcccttctcg 20400 gccggcaacgccacaacata aagaagcaag caacatcaac aacagctgcc gccatgggct 20460 ccagtgagcaggaactgaaa gccattgtca aagatcttgg ttgtgggcca tattttttgg 20520 gcacctatgacaagcgcttt ccaggctttg tttctccaca caagctcgcc tgcgccatag 20580 tcaatacggccggtcgcgag actgggggcg tacactggat ggcctttgcc tggaacccgc 20640 actcaaaaacatgctacctc tttgagccct ttggcttttc tgaccagcga ctcaagcagg 20700 tttaccagtttgagtacgag tcactcctgc gccgtagcgc cattgcttct tcccccgacc 20760 gctgtataacgctggaaaag tccacccaaa gcgtacaggg gcccaactcg gccgcctgtg 20820 gactattctgctgcatgttt ctccacgcct ttgccaactg gccccaaact cccatggatc 20880 acaaccccaccatgaacctt attaccgggg tacccaactc catgctcaac agtccccagg 20940 tacagcccaccctgcgtcgc aaccaggaac agctctacag cttcctggag cgccactcgc 21000 cctacttccgcagccacagt gcgcagatta ggagcgccac ttctttttgt cacttgaaaa 21060 acatgtaaaaataatgtact agagacactt tcaataaagg caaatgcttt tatttgtaca 21120 ctctcgggtgattatttacc cccacccttg ccgtctgcgc cgtttaaaaa tcaaaggggt 21180 tctgccgcgcatcgctatgc gccactggca gggacacgtt gcgatactgg tgtttagtgc 21240 tccacttaaactcaggcaca accatccgcg gcagctcggt gaagttttca ctccacaggc 21300 tgcgcaccatcaccaacgcg tttagcaggt cgggcgccga tatcttgaag tcgcagttgg 21360 ggcctccgccctgcgcgcgc gagttgcgat acacagggtt gcagcactgg aacactatca 21420 gcgccgggtggtgcacgctg gccagcacgc tcttgtcgga gatcagatcc gcgtccaggt 21480 cctccgcgttgctcagggcg aacggagtca actttggtag ctgccttccc aaaaagggcg 21540 cgtgcccaggctttgagttg cactcgcacc gtagtggcat caaaaggtga ccgtgcccgg 21600 tctgggcgttaggatacagc gcctgcataa aagccttgat ctgcttaaaa gccacctgag 21660 cctttgcgccttcagagaag aacatgccgc aagacttgcc ggaaaactga ttggccggac 21720 aggccgcgtcgtgcacgcag caccttgcgt cggtgttgga gatctgcacc acatttcggc 21780 cccaccggttcttcacgatc ttggccttgc tagactgctc cttcagcgcg cgctgcccgt 21840 tttcgctcgtcacatccatt tcaatcacgt gctccttatt tatcataatg cttccgtgta 21900 gacacttaagctcgccttcg atctcagcgc agcggtgcag ccacaacgcg cagcccgtgg 21960 gctcgtgatgcttgtaggtc acctctgcaa acgactgcag gtacgcctgc aggaatcgcc 22020 ccatcatcgtcacaaaggtc ttgttgctgg tgaaggtcag ctgcaacccg cggtgctcct 22080 cgttcagccaggtcttgcat acggccgcca gagcttccac ttggtcaggc agtagtttga 22140 agttcgcctttagatcgtta tccacgtggt acttgtccat cagcgcgcgc gcagcctcca 22200 tgcccttctcccacgcagac acgatcggca cactcagcgg gttcatcacc gtaatttcac 22260 tttccgcttcgctgggctct tcctcttcct cttgcgtccg cataccacgc gccactgggt 22320 cgtcttcattcagccgccgc actgtgcgct tacctccttt gccatgcttg attagcaccg 22380 gtgggttgctgaaacccacc atttgtagcg ccacatcttc tctttcttcc tcgctgtcca 22440 cgattacctctggtgatggc gggcgctcgg gcttgggaga agggcgcttc tttttcttct 22500 tgggcgcaatggccaaatcc gccgccgagg tcgatggccg cgggctgggt gtgcgcggca 22560 ccagcgcgtcttgtgatgag tcttcctcgt cctcggactc gatacgccgc ctcatccgct 22620 tttttgggggcgcccgggga ggcggcggcg acggggacgg ggacgacacg tcctccatgg 22680 ttgggggacgtcgcgccgca ccgcgtccgc gctcgggggt ggtttcgcgc tgctcctctt 22740 cccgactggccatttccttc tcctataggc agaaaaagat catggagtca gtcgagaaga 22800 aggacagcctaaccgccccc tctgagttcg ccaccaccgc ctccaccgat gccgccaacg 22860 cgcctaccaccttccccgtc gaggcacccc cgcttgagga ggaggaagtg attatcgagc 22920 aggacccaggttttgtaagc gaagacgacg aggaccgctc agtaccaaca gaggataaaa 22980 agcaagaccaggacaacgca gaggcaaacg aggaacaagt cgggcggggg gacgaaaggc 23040 atggcgactacctagatgtg ggagacgacg tgctgttgaa gcatctgcag cgccagtgcg 23100 ccattatctgcgacgcgttg caagagcgca gcgatgtgcc cctcgccata gcggatgtca 23160 gccttgcctacgaacgccac ctattctcac cgcgcgtacc ccccaaacgc caagaaaacg 23220 gcacatgcgagcccaacccg cgcctcaact tctaccccgt atttgccgtg ccagaggtgc 23280 ttgccacctatcacatcttt ttccaaaact gcaagatacc cctatcctgc cgtgccaacc 23340 gcagccgagcggacaagcag ctggccttgc ggcagggcgc tgtcatacct gatatcgcct 23400 cgctcaacgaagtgccaaaa atctttgagg gtcttggacg cgacgagaag cgcgcggcaa 23460 acgctctgcaacaggaaaac agcgaaaatg aaagtcactc tggagtgttg gtggaactcg 23520 agggtgacaacgcgcgccta gccgtactaa aacgcagcat cgaggtcacc cactttgcct 23580 acccggcacttaacctaccc cccaaggtca tgagcacagt catgagtgag ctgatcgtgc 23640 gccgtgcgcagcccctggag agggatgcaa atttgcaaga acaaacagag gagggcctac 23700 ccgcagttggcgacgagcag ctagcgcgct ggcttcaaac gcgcgagcct gccgacttgg 23760 aggagcgacgcaaactaatg atggccgcag tgctcgttac cgtggagctt gagtgcatgc 23820 agcggttctttgctgacccg gagatgcagc gcaagctaga ggaaacattg cactacacct 23880 ttcgacagggctacgtacgc caggcctgca agatctccaa cgtggagctc tgcaacctgg 23940 tctcctaccttggaattttg cacgaaaacc gccttgggca aaacgtgctt cattccacgc 24000 tcaagggcgaggcgcgccgc gactacgtcc gcgactgcgt ttacttattt ctatgctaca 24060 cctggcagacggccatgggc gtttggcagc agtgcttgga ggagtgcaac ctcaaggagc 24120 tgcagaaactgctaaagcaa aacttgaagg acctatggac ggccttcaac gagcgctccg 24180 tggccgcgcacctggcggac atcattttcc ccgaacgcct gcttaaaacc ctgcaacagg 24240 gtctgccagacttcaccagt caaagcatgt tgcagaactt taggaacttt atcctagagc 24300 gctcaggaatcttgcccgcc acctgctgtg cacttcctag cgactttgtg cccattaagt 24360 accgcgaatgccctccgccg ctttggggcc actgctacct tctgcagcta gccaactacc 24420 ttgcctaccactctgacata atggaagacg tgagcggtga cggtctactg gagtgtcact 24480 gtcgctgcaacctatgcacc ccgcaccgct ccctggtttg caattcgcag ctgcttaacg 24540 aaagtcaaattatcggtacc tttgagctgc agggtccctc gcctgacgaa aagtccgcgg 24600 ctccggggttgaaactcact ccggggctgt ggacgtcggc ttaccttcgc aaatttgtac 24660 ctgaggactaccacgcccac gagattaggt tctacgaaga ccaatcccgc ccgccaaatg 24720 cggagcttaccgcctgcgtc attacccagg gccacattct tggccaattg caagccatca 24780 acaaagcccgccaagagttt ctgctacgaa agggacgggg ggtttacttg gacccccagt 24840 ccggcgaggagctcaaccca atccccccgc cgccgcagcc ctatcagcag cagccgcggg 24900 cccttgcttcccaggatggc acccaaaaag aagctgcagc tgccgccgcc acccacggac 24960 gaggaggaatactgggacag tcaggcagag gaggttttgg acgaggagga ggaggacatg 25020 atggaagactgggagagcct agacgaggaa gcttccgagg tcgaagaggt gtcagacgaa 25080 acaccgtcaccctcggtcgc attcccctcg ccggcgcccc agaaatcggc aaccggttcc 25140 agcatggctacaacctccgc tcctcaggcg ccgccggcac tgcccgttcg ccgacccaac 25200 cgtagatgggacaccactgg aaccagggcc ggtaagtcca agcagccgcc gccgttagcc 25260 caagagcaacaacagcgcca aggctaccgc tcatggcgcg ggcacaagaa cgccatagtt 25320 gcttgcttgcaagactgtgg gggcaacatc tccttcgccc gccgctttct tctctaccat 25380 cacggcgtggccttcccccg taacatcctg cattactacc gtcatctcta cagcccatac 25440 tgcaccggcggcagcggcag cggcagcaac agcagcggcc acacagaagc aaaggcgacc 25500 ggatagcaagactctgacaa agcccaagaa atccacagcg gcggcagcag caggaggagg 25560 agcgctgcgtctggcgccca acgaacccgt atcgacccgc gagcttagaa acaggatttt 25620 tcccactctgtatgctatat ttcaacagag caggggccaa gaacaagagc tgaaaataaa 25680 aaacaggtctctgcgatccc tcacccgcag ctgcctgtat cacaaaagcg aagatcagct 25740 tcggcgcacgctggaagacg cggaggctct cttcagtaaa tactgcgcgc tgactcttaa 25800 ggactagtttcgcgcccttt ctcaaattta agcgcgaaaa ctacgtcatc tccagcggcc 25860 acacccggcgccagcacctg tcgtcagcgc cattatgagc aaggaaattc ccacgcccta 25920 catgtggagttaccagccac aaatgggact tgcggctgga gctgcccaag actactcaac 25980 ccgaataaactacatgagcg cgggacccca catgatatcc cgggtcaacg gaatccgcgc 26040 ccaccgaaaccgaattctct tggaacaggc ggctattacc accacacctc gtaataacct 26100 taatccccgtagttggcccg ctgccctggt gtaccaggaa agtcccgctc ccaccactgt 26160 ggtacttcccagagacgccc aggccgaagt tcagatgact aactcagggg cgcagcttgc 26220 gggcggctttcgtcacaggg tgcggtcgcc cgggcagggt ataactcacc tgacaatcag 26280 agggcgaggtattcagctca acgacgagtc ggtgagctcc tcgcttggtc tccgtccgga 26340 cgggacatttcagatcggcg gcgccggccg tccttcattc acgcctcgtc aggcaatcct 26400 aactctgcagacctcgtcct ctgagccgcg ctctggaggc attggaactc tgcaatttat 26460 tgaggagtttgtgccatcgg tctactttaa ccccttctcg ggacctcccg gccactatcc 26520 ggatcaatttattcctaact ttgacgcggt aaaggactcg gcggacggct acgactgaat 26580 gttaagtggagaggcagagc aactgcgcct gaaacacctg gtccactgtc gccgccacaa 26640 gtgctttgcccgcgactccg gtgagttttg ctactttgaa ttgcccgagg atcatatcga 26700 gggcccggcgcacggcgtcc ggcttaccgc ccagggagag cttgcccgta gcctgattcg 26760 ggagtttacccagcgccccc tgctagttga gcgggacagg ggaccctgtg ttctcactgt 26820 gatttgcaactgtcctaacc ttggattaca tcaagatctt tgttgccatc tctgtgctga 26880 gtataataaatacagaaatt aaaatatact ggggctccta tcgccatcct gtaaacgcca 26940 ccgtcttcacccgcccaagc aaaccaaggc gaaccttacc tggtactttt aacatctctc 27000 cctctgtgatttacaacagt ttcaacccag acggagtgag tctacgagag aacctctccg 27060 agctcagctactccatcaga aaaaacacca ccctccttac ctgccgggaa cgtacgagtg 27120 cgtcaccggccgctgcacca cacctaccgc ctgaccgtaa accagacttt ttccggacag 27180 acctcaataactctgtttac cagaacagga ggtgagctta gaaaaccctt agggtattag 27240 gccaaaggcgcagctactgt ggggtttatg aacaattcaa gcaactctac gggctattct 27300 aattcaggtttctctagaaa tggacggaat tattacagag cagcgcctgc tagaaagacg 27360 cagggcagcggccgagcaac agcgcatgaa tcaagagctc caagacatgg ttaacttgca 27420 ccagtgcaaaaggggtatct tttgtctggt aaagcaggcc aaagtcacct acgacagtaa 27480 taccaccggacaccgcctta gctacaagtt gccaaccaag cgtcagaaat tggtggtcat 27540 ggtgggagaaaagcccatta ccataactca gcactcggta gaaaccgaag gctgcattca 27600 ctcaccttgtcaaggacctg aggatctctg cacccttatt aagaccctgt gcggtctcaa 27660 agatcttattccctttaact aataaaaaaa aataataaag catcacttac ttaaaatcag 27720 ttagcaaatttctgtccagt ttattcagca gcacctcctt gccctcctcc cagctctggt 27780 attgcagcttcctcctggct gcaaactttc tccacaatct aaatggaatg tcagtttcct 27840 cctgttcctgtccatccgca cccactatct tcatgttgtt gcagatgaag cgcgcaagac 27900 cgtctgaagataccttcaac cccgtgtatc catatgacac ggaaaccggt cctccaactg 27960 tgccttttcttactcctccc tttgtatccc ccaatgggtt tcaagagagt ccccctgggg 28020 tactctctttgcgcctatcc gaacctctag ttacctccaa tggcatgctt gcgctcaaaa 28080 tgggcaacggcctctctctg gacgaggccg gcaaccttac ctcccaaaat gtaaccactg 28140 tgagcccacctctcaaaaaa accaagtcaa acataaacct ggaaatatct gcacccctca 28200 cagttacctcagaagcccta actgtggctg ccgccgcacc tctaatggtc gcgggcaaca 28260 cactcaccatgcaatcacag gccccgctaa ccgtgcacga ctccaaactt agcattgcca 28320 cccaaggacccctcacagtg tcagaaggaa agctagccct gcaaacatca ggccccctca 28380 ccaccaccgatagcagtacc cttactatca ctgcctcacc ccctctaact actgccactg 28440 gtagcttgggcattgacttg aaagagccca tttatacaca aaatggaaaa ctaggactaa 28500 agtacggggctcctttgcat gtaacagacg acctaaacac tttgaccgta gcaactggtc 28560 caggtgtgactattaataat acttccttgc aaactaaagt tactggagcc ttgggttttg 28620 attcacaaggcaatatgcaa cttaatgtag caggaggact aaggattgat tctcaaaaca 28680 gacgccttatacttgatgtt agttatccgt ttgatgctca aaaccaacta aatctaagac 28740 taggacagggccctcttttt ataaactcag cccacaactt ggatattaac tacaacaaag 28800 gcctttacttgtttacagct tcaaacaatt ccaaaaagct tgaggttaac ctaagcactg 28860 ccaaggggttgatgtttgac gctacagcca tagccattaa tgcaggagat gggcttgaat 28920 ttggttcacctaatgcacca aacacaaatc ccctcaaaac aaaaattggc catggcctag 28980 aatttgattcaaacaaggct atggttccta aactaggaac tggccttagt tttgacagca 29040 caggtgccattacagtagga aacaaaaata atgataagct aactttgtgg accacaccag 29100 ctccatctcctaactgtaga ctaaatgcag agaaagatgc taaactcact ttggtcttaa 29160 caaaatgtggcagtcaaata cttgctacag tttcagtttt ggctgttaaa ggcagtttgg 29220 ctccaatatctggaacagtt caaagtgctc atcttattat aagatttgac gaaaatggag 29280 tgctactaaacaattccttc ctggacccag aatattggaa ctttagaaat ggagatctta 29340 ctgaaggcacagcctataca aacgctgttg gatttatgcc taacctatca gcttatccaa 29400 aatctcacggtaaaactgcc aaaagtaaca ttgtcagtca agtttactta aacggagaca 29460 aaactaaacctgtaacacta accattacac taaacggtac acaggaaaca ggagacacaa 29520 ctccaagtgcatactctatg tcattttcat gggactggtc tggccacaac tacattaatg 29580 aaatatttgccacatcctct tacacttttt catacattgc ccaagaataa agaatcgttt 29640 gtgttatgtttcaacgtgtt tatttttcaa ttgcagaaaa tttcgaatca tttttcattc 29700 agtagtatagccccaccacc acatagctta tacagatcac cgtaccttaa tcaaactcac 29760 agaaccctagtattcaacct gccacctccc tcccaacaca cagagtacac agtcctttct 29820 ccccggctggccttaaaaag catcatatca tgggtaacag acatattctt aggtgttata 29880 ttccacacggtttcctgtcg agccaaacgc tcatcagtga tattaataaa ctccccgggc 29940 agctcacttaagttcatgtc gctgtccagc tgctgagcca caggctgctg tccaacttgc 30000 ggttgcttaacgggcggcga aggagaagtc cacgcctaca tgggggtaga gtcataatcg 30060 tgcatcaggatagggcggtg gtgctgcagc agcgcgcgaa taaactgctg ccgccgccgc 30120 tccgtcctgcaggaatacaa catggcagtg gtctcctcag cgatgattcg caccgcccgc 30180 agcataaggcgccttgtcct ccgggcacag cagcgcaccc tgatctcact taaatcagca 30240 cagtaactgcagcacagcac cacaatattg ttcaaaatcc cacagtgcaa ggcgctgtat 30300 ccaaagctcatggcggggac cacagaaccc acgtggccat cataccacaa gcgcaggtag 30360 attaagtggcgacccctcat aaacacgctg gacataaaca ttacctcttt tggcatgttg 30420 taattcaccacctcccggta ccatataaac ctctgattaa acatggcgcc atccaccacc 30480 atcctaaaccagctggccaa aacctgcccg ccggctatac actgcaggga accgggactg 30540 gaacaatgacagtggagagc ccaggactcg taaccatgga tcatcatgct cgtcatgata 30600 tcaatgttggcacaacacag gcacacgtgc atacacttcc tcaggattac aagctcctcc 30660 cgcgttagaaccatatccca gggaacaacc cattcctgaa tcagcgtaaa tcccacactg 30720 cagggaagacctcgcacgta actcacgttg tgcattgtca aagtgttaca ttcgggcagc 30780 agcggatgatcctccagtat ggtagcgcgg gtttctgtct caaaaggagg tagacgatcc 30840 ctactgtacggagtgcgccg agacaaccga gatcgtgttg gtcgtagtgt catgccaaat 30900 ggaacgccggacgtagtcat atttcctgaa gcaaaaccag gtgcgggcgt gacaaacaga 30960 tctgcgtctccggtctcgcc gcttagatcg ctctgtgtag tagttgtagt atatccactc 31020 tctcaaagcatccaggcgcc ccctggcttc gggttctatg taaactcctt catgcgccgc 31080 tgccctgataacatccacca ccgcagaata agccacaccc agccaaccta cacattcgtt 31140 ctgcgagtcacacacgggag gagcgggaag agctggaaga accatgtttt tttttttatt 31200 ccaaaagattatccaaaacc tcaaaatgaa gatctattaa gtgaacgcgc tcccctccgg 31260 tggcgtggtcaaactctaca gccaaagaac agataatggc atttgtaaga tgttgcacaa 31320 tggcttccaaaaggcaaacg gccctcacgt ccaagtggac gtaaaggcta aacccttcag 31380 ggtgaatctcctctataaac attccagcac cttcaaccat gcccaaataa ttctcatctc 31440 gccaccttctcaatatatct ctaagcaaat cccgaatatt aagtccggcc attgtaaaaa 31500 tctgctccagagcgccctcc accttcagcc tcaagcagcg aatcatgatt gcaaaaattc 31560 aggttcctcacagacctgta taagattcaa aagcggaaca ttaacaaaaa taccgcgatc 31620 ccgtaggtcccttcgcaggg ccagctgaac ataatcgtgc aggtctgcac ggaccagcgc 31680 ggccacttccccgccaggaa ccttgacaaa agaacccaca ctgattatga cacgcatact 31740 cggagctatgctaaccagcg tagccccgat gtaagctttg ttgcatgggc ggcgatataa 31800 aatgcaaggtgctgctcaaa aaatcaggca aagcctcgcg caaaaaagaa agcacatcgt 31860 agtcatgctcatgcagataa aggcaggtaa gctccggaac caccacagaa aaagacacca 31920 tttttctctcaaacatgtct gcgggtttct gcataaacac aaaataaaat aacaaaaaaa 31980 catttaaacattagaagcct gtcttacaac aggaaaaaca acccttataa gcataagacg 32040 gactacggccatgccggcgt gaccgtaaaa aaactggtca ccgtgattaa aaagcaccac 32100 cgacagctcctcggtcatgt ccggagtcat aatgtaagac tcggtaaaca catcaggttg 32160 attcacatcggtcagtgcta aaaagcgacc gaaatagccc gggggaatac atacccgcag 32220 gcgtagagacaacattacag cccccatagg aggtataaca aaattaatag gagagaaaaa 32280 cacataaacacctgaaaaac cctcctgcct aggcaaaata gcaccctccc gctccagaac 32340 aacatacagcgcttccacag cggcagccat aacagtcagc cttaccagta aaaaagaaaa 32400 cctattaaaaaaacaccact cgacacggca ccagctcaat cagtcacagt gtaaaaaagg 32460 gccaagtgcagagcgagtat atataggact aaaaaatgac gtaacggtta aagtccacaa 32520 aaaacacccagaaaaccgca cgcgaaccta cgcccagaaa cgaaagccaa aaaacccaca 32580 acttcctcaaatcgtcactt ccgttttccc acgttacgtc acttcccatt ttaagaaaac 32640 tacaattcccaacacataca agttactccg ccctaaaacc tacgtcaccc gccccgttcc 32700 cacgccccgcgccacgtcac aaactccacc ccctcattat catattggct tcaatccaaa 32760 ataaggtatattattgatga tgttaattaa tttaaatccg catgcgatat cgagctctcc 32820 cgggaattcggatctgcgac gcgaggctgg atggccttcc ccattatgat tcttctcgct 32880 tccggcggcatcgggatgcc cgcgttgcag gccatgctgt ccaggcaggt agatgacgac 32940 catcagggacagcttcacgg ccagcaaaag gccaggaacc gtaaaaaggc cgcgttgctg 33000 gcgtttttccataggctccg cccccctgac gagcatcaca aaaatcgacg ctcaagtcag 33060 aggtggcgaaacccgacagg actataaaga taccaggcgt ttccccctgg aagctccctc 33120 gtgcgctctcctgttccgac cctgccgctt accggatacc tgtccgcctt tctcccttcg 33180 ggaagcgtggcgctttctca atgctcacgc tgtaggtatc tcagttcggt gtaggtcgtt 33240 cgctccaagctgggctgtgt gcacgaaccc cccgttcagc ccgaccgctg cgccttatcc 33300 ggtaactatcgtcttgagtc caacccggta agacacgact tatcgccact ggcagcagcc 33360 actggtaacaggattagcag agcgaggtat gtaggcggtg ctacagagtt cttgaagtgg 33420 tggcctaactacggctacac tagaaggaca gtatttggta tctgcgctct gctgaagcca 33480 gttaccttcggaaaaagagt tggtagctct tgatccggca aacaaaccac cgctggtagc 33540 ggtggtttttttgtttgcaa gcagcagatt acgcgcagaa aaaaaggatc tcaagaagat 33600 cctttgatcttttctacggg gtctgacgct cagtggaacg aaaactcacg ttaagggatt 33660 ttggtcatgagattatcaaa aaggatcttc acctagatcc ttttaaatca atctaaagta 33720 tatatgagtaaacttggtct gacagttacc aatgcttaat cagtgaggca cctatctcag 33780 cgatctgtctatttcgttca tccatagttg cctgactccc cgtcgtgtag ataactacga 33840 tacgggagggcttaccatct ggccccagtg ctgcaatgat accgcgagac ccacgctcac 33900 cggctccagatttatcagca ataaaccagc cagccggaag ggccgagcgc agaagtggtc 33960 ctgcaactttatccgcctcc atccagtcta ttaattgttg ccgggaagct agagtaagta 34020 gttcgccagttaatagtttg cgcaacgttg ttgccattgn tgcaggcatc gtggtgtcac 34080 gctcgtcgtttggtatggct tcattcagct ccggttccca acgatcaagg cgagttacat 34140 gatcccccatgttgtgcaaa aaagcggtta gctccttcgg tcctccgatc gttgtcagaa 34200 gtaagttggccgcagtgtta tcactcatgg ttatggcagc actgcataat tctcttactg 34260 tcatgccatccgtaagatgc ttttctgtga ctggtgagta ctcaaccaag tcattctgag 34320 aatagtgtatgcggcgaccg agttgctctt gcccggcgtc aacacgggat aataccgcgc 34380 cacatagcagaactttaaaa gtgctcatca ttggaaaacg ttcttcgggg cgaaaactct 34440 caaggatcttaccgctgttg agatccagtt cgatgtaacc cactcgtgca cccaactgat 34500 cttcagcatcttttactttc accagcgttt ctgggtgagc aaaaacagga aggcaaaatg 34560 ccgcaaaaaagggaataagg gcgacacgga aatgttgaat actcatactc ttcctttttc 34620 aatattattgaagcatttat cagggttatt gtctcatgag cggatacata tttgaatgta 34680 tttagaaaaataaacaaata ggggttccgc gcacatttcc ccgaaaagtg ccacctgacg 34740 tctaagaaaccattattatc atgacattaa cctataaaaa taggcgtatc acgaggccct 34800 ttcgtcttcaaggatccgaa ttcccgggag agctcgatat cgcatgcgga tttaaattaa 34860 ttaa 3486488 37567 DNA Artificial Sequence pAd/CMV/V5-GW/lacZ.PL-DEST 88catcatcaat aatatacctt attttggatt gaagccaata tgataatgag ggggtggagt 60ttgtgacgtg gcgcggggcg tgggaacggg gcgggtgacg tagtagtgtg gcggaagtgt 120gatgttgcaa gtgtggcgga acacatgtaa gcgacggatg tggcaaaagt gacgtttttg 180gtgtgcgccg gtgtacacag gaagtgacaa ttttcgcgcg gttttaggcg gatgttgtag 240taaatttggg cgtaaccgag taagatttgg ccattttcgc gggaaaactg aataagagga 300agtgaaatct gaataatttt gtgttactca tagcgcgtaa tatttgtcta gggccgcggg 360gactttgacc gtttacgtgg agactcgccc aggtgttttt ctcaggtgtt ttccgcgttc 420cgggtcaaag ttggcgtttt attattatag tcagtcgaag cttggatccg gtacctctag 480aattctcgag cggccgctag cgacatcgga tctcccgatc ccctatggtc gactctcagt 540acaatctgct ctgatgccgc atagttaagc cagtatctgc tccctgcttg tgtgttggag 600gtcgctgagt agtgcgcgag caaaatttaa gctacaacaa ggcaaggctt gaccgacaat 660tgcatgaaga atctgcttag ggttaggcgt tttgcgctgc ttcgcgatgt acgggccaga 720tatacgcgtt gacattgatt attgactagt tattaatagt aatcaattac ggggtcatta 780gttcatagcc catatatgga gttccgcgtt acataactta cggtaaatgg cccgcctggc 840tgaccgccca acgacccccg cccattgacg tcaataatga cgtatgttcc catagtaacg 900ccaataggga ctttccattg acgtcaatgg gtggactatt tacggtaaac tgcccacttg 960gcagtacatc aagtgtatca tatgccaagt acgcccccta ttgacgtcaa tgacggtaaa 1020tggcccgcct ggcattatgc ccagtacatg accttatggg actttcctac ttggcagtac 1080atctacgtat tagtcatcgc tattaccatg gtgatgcggt tttggcagta catcaatggg 1140cgtggatagc ggtttgactc acggggattt ccaagtctcc accccattga cgtcaatggg 1200agtttgtttt ggcaccaaaa tcaacgggac tttccaaaat gtcgtaacaa ctccgcccca 1260ttgacgcaaa tgggcggtag gcgtgtacgg tgggaggtct atataagcag agctctctgg 1320ctaactagag aacccactgc ttactggctt atcgaaatta atacgactca ctatagggag 1380acccaagctg gctagttaag ctatcaacaa gtttgtacaa aaaagcaggc tccgcggccg 1440cccccttcac catgatagat cccgtcgttt tacaacgtcg tgactgggaa aaccctggcg 1500ttacccaact taatcgcctt gcagcacatc cccctttcgc cagctggcgt aatagcgaag 1560aggcccgcac cgatcgccct tcccaacagt tgcgcagcct gaatggcgaa tggcgctttg 1620cctggtttcc ggcaccagaa gcggtgccgg aaagctggct ggagtgcgat cttcctgagg 1680ccgatactgt cgtcgtcccc tcaaactggc agatgcacgg ttacgatgcg cccatctaca 1740ccaacgtaac ctatcccatt acggtcaatc cgccgtttgt tcccacggag aatccgacgg 1800gttgttactc gctcacattt aatgttgatg aaagctggct acaggaaggc cagacgcgaa 1860ttatttttga tggcgttaac tcggcgtttc atctgtggtg caacgggcgc tgggtcggtt 1920acggccagga cagtcgtttg ccgtctgaat ttgacctgag cgcattttta cgcgccggag 1980aaaaccgcct cgcggtgatg gtgctgcgtt ggagtgacgg cagttatctg gaagatcagg 2040atatgtggcg gatgagcggc attttccgtg acgtctcgtt gctgcataaa ccgactacac 2100aaatcagcga tttccatgtt gccactcgct ttaatgatga tttcagccgc gctgtactgg 2160aggctgaagt tcagatgtgc ggcgagttgc gtgactacct acgggtaaca gtttctttat 2220ggcagggtga aacgcaggtc gccagcggca ccgcgccttt cggcggtgaa attatcgatg 2280agcgtggtgg ttatgccgat cgcgtcacac tacgtctgaa cgtcgaaaac ccgaaactgt 2340ggagcgccga aatcccgaat ctctatcgtg cggtggttga actgcacacc gccgacggca 2400cgctgattga agcagaagcc tgcgatgtcg gtttccgcga ggtgcggatt gaaaatggtc 2460tgctgctgct gaacggcaag ccgttgctga ttcgaggcgt taaccgtcac gagcatcatc 2520ctctgcatgg tcaggtcatg gatgagcaga cgatggtgca ggatatcctg ctgatgaagc 2580agaacaactt taacgccgtg cgctgttcgc attatccgaa ccatccgctg tggtacacgc 2640tgtgcgaccg ctacggcctg tatgtggtgg atgaagccaa tattgaaacc cacggcatgg 2700tgccaatgaa tcgtctgacc gatgatccgc gctggctacc ggcgatgagc gaacgcgtaa 2760cgcgaatggt gcagcgcgat cgtaatcacc cgagtgtgat catctggtcg ctggggaatg 2820aatcaggcca cggcgctaat cacgacgcgc tgtatcgctg gatcaaatct gtcgatcctt 2880cccgcccggt gcagtatgaa ggcggcggag ccgacaccac ggccaccgat attatttgcc 2940cgatgtacgc gcgcgtggat gaagaccagc ccttcccggc tgtgccgaaa tggtccatca 3000aaaaatggct ttcgctacct ggagagacgc gcccgctgat cctttgcgaa tacgcccacg 3060cgatgggtaa cagtcttggc ggtttcgcta aatactggca ggcgtttcgt cagtatcccc 3120gtttacaggg cggcttcgtc tgggactggg tggatcagtc gctgattaaa tatgatgaaa 3180acggcaaccc gtggtcggct tacggcggtg attttggcga tacgccgaac gatcgccagt 3240tctgtatgaa cggtctggtc tttgccgacc gcacgccgca tccagcgctg acggaagcaa 3300aacaccagca gcagtttttc cagttccgtt tatccgggca aaccatcgaa gtgaccagcg 3360aatacctgtt ccgtcatagc gataacgagc tcctgcactg gatggtggcg ctggatggta 3420agccgctggc aagcggtgaa gtgcctctgg atgtcgctcc acaaggtaaa cagttgattg 3480aactgcctga actaccgcag ccggagagcg ccgggcaact ctggctcaca gtacgcgtag 3540tgcaaccgaa cgcgaccgca tggtcagaag ccgggcacat cagcgcctgg cagcagtggc 3600gtctggcgga aaacctcagt gtgacgctcc ccgccgcgtc ccacgccatc ccgcatctga 3660ccaccagcga aatggatttt tgcatcgagc tgggtaataa gcgttggcaa tttaaccgcc 3720agtcaggctt tctttcacag atgtggattg gcgataaaaa acaactgctg acgccgctgc 3780gcgatcagtt cacccgtgca ccgctggata acgacattgg cgtaagtgaa gcgacccgca 3840ttgaccctaa cgcctgggtc gaacgctgga aggcggcggg ccattaccag gccgaagcag 3900cgttgttgca gtgcacggca gatacacttg ctgatgcggt gctgattacg accgctcacg 3960cgtggcagca tcaggggaaa accttattta tcagccggaa aacctaccgg attgatggta 4020gtggtcaaat ggcgattacc gttgatgttg aagtggcgag cgatacaccg catccggcgc 4080ggattggcct gaactgccag ctggcgcagg tagcagagcg ggtaaactgg ctcggattag 4140ggccgcaaga aaactatccc gaccgcctta ctgccgcctg ttttgaccgc tgggatctgc 4200cattgtcaga catgtatacc ccgtacgtct tcccgagcga aaacggtctg cgctgcggga 4260cgcgcgaatt gaattatggc ccacaccagt ggcgcggcga cttccagttc aacatcagcc 4320gctacagtca acagcaactg atggaaacca gccatcgcca tctgctgcac gcggaagaag 4380gcacatggct gaatatcgac ggtttccata tggggattgg tggcgacgac tcctggagcc 4440cgtcagtatc ggcggagttc cagctgagcg ccggtcgcta ccattaccag ttggtctggt 4500gtcaaaaaac taagggtggg cgcgccgacc cagctttctt gtacaaagtg gttgatctag 4560agggcccgcg gttcgaaggt aagcctatcc ctaaccctct cctcggtctc gattctacgc 4620gtaccggtta gtaatgagtt taaacggggg aggctaactg aaacacggaa ggagacaata 4680ccggaaggaa cccgcgctat gacggcaata aaaagacaga ataaaacgca cgggtgttgg 4740gtcgtttgtt cataaacgcg gggttcggtc ccagggctgg cactctgtcg ataccccacc 4800gagaccccat tggggccaat acgcccgcgt ttcttccttt tccccacccc accccccaag 4860ttcgggtgaa ggcccagggc tcgcagccaa cgtcggggcg gcaggccctg ccatagcaga 4920tccgattcga cagatcactg aaatgtgtgg gcgtggctta agggtgggaa agaatatata 4980aggtgggggt cttatgtagt tttgtatctg ttttgcagca gccgccgccg ccatgagcac 5040caactcgttt gatggaagca ttgtgagctc atatttgaca acgcgcatgc ccccatgggc 5100cggggtgcgt cagaatgtga tgggctccag cattgatggt cgccccgtcc tgcccgcaaa 5160ctctactacc ttgacctacg agaccgtgtc tggaacgccg ttggagactg cagcctccgc 5220cgccgcttca gccgctgcag ccaccgcccg cgggattgtg actgactttg ctttcctgag 5280cccgcttgca agcagtgcag cttcccgttc atccgcccgc gatgacaagt tgacggctct 5340tttggcacaa ttggattctt tgacccggga acttaatgtc gtttctcagc agctgttgga 5400tctgcgccag caggtttctg ccctgaaggc ttcctcccct cccaatgcgg tttaaaacat 5460aaataaaaaa ccagactctg tttggatttg gatcaagcaa gtgtcttgct gtctttattt 5520aggggttttg cgcgcgcggt aggcccggga ccagcggtct cggtcgttga gggtcctgtg 5580tattttttcc aggacgtggt aaaggtgact ctggatgttc agatacatgg gcataagccc 5640gtctctgggg tggaggtagc accactgcag agcttcatgc tgcggggtgg tgttgtagat 5700gatccagtcg tagcaggagc gctgggcgtg gtgcctaaaa atgtctttca gtagcaagct 5760gattgccagg ggcaggccct tggtgtaagt gtttacaaag cggttaagct gggatgggtg 5820catacgtggg gatatgagat gcatcttgga ctgtattttt aggttggcta tgttcccagc 5880catatccctc cggggattca tgttgtgcag aaccaccagc acagtgtatc cggtgcactt 5940gggaaatttg tcatgtagct tagaaggaaa tgcgtggaag aacttggaga cgcccttgtg 6000acctccaaga ttttccatgc attcgtccat aatgatggca atgggcccac gggcggcggc 6060ctgggcgaag atatttctgg gatcactaac gtcatagttg tgttccagga tgagatcgtc 6120ataggccatt tttacaaagc gcgggcggag ggtgccagac tgcggtataa tggttccatc 6180cggcccaggg gcgtagttac cctcacagat ttgcatttcc cacgctttga gttcagatgg 6240ggggatcatg tctacctgcg gggcgatgaa gaaaacggtt tccggggtag gggagatcag 6300ctgggaagaa agcaggttcc tgagcagctg cgacttaccg cagccggtgg gcccgtaaat 6360cacacctatt accgggtgca actggtagtt aagagagctg cagctgccgt catccctgag 6420caggggggcc acttcgttaa gcatgtccct gactcgcatg ttttccctga ccaaatccgc 6480cagaaggcgc tcgccgccca gcgatagcag ttcttgcaag gaagcaaagt ttttcaacgg 6540tttgagaccg tccgccgtag gcatgctttt gagcgtttga ccaagcagtt ccaggcggtc 6600ccacagctcg gtcacctgct ctacggcatc tcgatccagc atatctcctc gtttcgcggg 6660ttggggcggc tttcgctgta cggcagtagt cggtgctcgt ccagacgggc cagggtcatg 6720tctttccacg ggcgcagggt cctcgtcagc gtagtctggg tcacggtgaa ggggtgcgct 6780ccgggctgcg cgctggccag ggtgcgcttg aggctggtcc tgctggtgct gaagcgctgc 6840cggtcttcgc cctgcgcgtc ggccaggtag catttgacca tggtgtcata gtccagcccc 6900tccgcggcgt ggcccttggc gcgcagcttg cccttggagg aggcgccgca cgaggggcag 6960tgcagacttt tgagggcgta gagcttgggc gcgagaaata ccgattccgg ggagtaggca 7020tccgcgccgc aggccccgca gacggtctcg cattccacga gccaggtgag ctctggccgt 7080tcggggtcaa aaaccaggtt tcccccatgc tttttgatgc gtttcttacc tctggtttcc 7140atgagccggt gtccacgctc ggtgacgaaa aggctgtccg tgtccccgta tacagacttg 7200agaggcctgt cctcgagcgg tgttccgcgg tcctcctcgt atagaaactc ggaccactct 7260gagacaaagg ctcgcgtcca ggccagcacg aaggaggcta agtgggaggg gtagcggtcg 7320ttgtccacta gggggtccac tcgctccagg gtgtgaagac acatgtcgcc ctcttcggca 7380tcaaggaagg tgattggttt gtaggtgtag gccacgtgac cgggtgttcc tgaagggggg 7440ctataaaagg gggtgggggc gcgttcgtcc tcactctctt ccgcatcgct gtctgcgagg 7500gccagctgtt ggggtgagta ctccctctga aaagcgggca tgacttctgc gctaagattg 7560tcagtttcca aaaacgagga ggatttgata ttcacctggc ccgcggtgat gcctttgagg 7620gtggccgcat ccatctggtc agaaaagaca atctttttgt tgtcaagctt ggtggcaaac 7680gacccgtaga gggcgttgga cagcaacttg gcgatggagc gcagggtttg gtttttgtcg 7740cgatcggcgc gctccttggc cgcgatgttt agctgcacgt attcgcgcgc aacgcaccgc 7800cattcgggaa agacggtggt gcgctcgtcg ggcaccaggt gcacgcgcca accgcggttg 7860tgcagggtga caaggtcaac gctggtggct acctctccgc gtaggcgctc gttggtccag 7920cagaggcggc cgcccttgcg cgagcagaat ggcggtaggg ggtctagctg cgtctcgtcc 7980ggggggtctg cgtccacggt aaagaccccg ggcagcaggc gcgcgtcgaa gtagtctatc 8040ttgcatcctt gcaagtctag cgcctgctgc catgcgcggg cggcaagcgc gcgctcgtat 8100gggttgagtg ggggacccca tggcatgggg tgggtgagcg cggaggcgta catgccgcaa 8160atgtcgtaaa cgtagagggg ctctctgagt attccaagat atgtagggta gcatcttcca 8220ccgcggatgc tggcgcgcac gtaatcgtat agttcgtgcg agggagcgag gaggtcggga 8280ccgaggttgc tacgggcggg ctgctctgct cggaagacta tctgcctgaa gatggcatgt 8340gagttggatg atatggttgg acgctggaag acgttgaagc tggcgtctgt gagacctacc 8400gcgtcacgca cgaaggaggc gtaggagtcg cgcagcttgt tgaccagctc ggcggtgacc 8460tgcacgtcta gggcgcagta gtccagggtt tccttgatga tgtcatactt atcctgtccc 8520ttttttttcc acagctcgcg gttgaggaca aactcttcgc ggtctttcca gtactcttgg 8580atcggaaacc cgtcggcctc cgaacggtaa gagcctagca tgtagaactg gttgacggcc 8640tggtaggcgc agcatccctt ttctacgggt agcgcgtatg cctgcgcggc cttccggagc 8700gaggtgtggg tgagcgcaaa ggtgtccctg accatgactt tgaggtactg gtatttgaag 8760tcagtgtcgt cgcatccgcc ctgctcccag agcaaaaagt ccgtgcgctt tttggaacgc 8820ggatttggca gggcgaaggt gacatcgttg aagagtatct ttcccgcgcg aggcataaag 8880ttgcgtgtga tgcggaaggg tcccggcacc tcggaacggt tgttaattac ctgggcggcg 8940agcacgatct cgtcaaagcc gttgatgttg tggcccacaa tgtaaagttc caagaagcgc 9000gggatgccct tgatggaagg caatttttta agttcctcgt aggtgagctc ttcaggggag 9060ctgagcccgt gctctgaaag ggcccagtct gcaagatgag ggttggaagc gacgaatgag 9120ctccacaggt cacgggccat tagcatttgc aggtggtcgc gaaaggtcct aaactggcga 9180cctatggcca ttttttctgg ggtgatgcag tagaaggtaa gcgggtcttg ttcccagcgg 9240tcccatccaa ggttcgcggc taggtctcgc gcggcagtca ctagaggctc atctccgccg 9300aacttcatga ccagcatgaa gggcacgagc tgcttcccaa aggcccccat ccaagtatag 9360gtctctacat cgtaggtgac aaagagacgc tcggtgcgag gatgcgagcc gatcgggaag 9420aactggatct cccgccacca attggaggag tggctattga tgtggtgaaa gtagaagtcc 9480ctgcgacggg ccgaacactc gtgctggctt ttgtaaaaac gtgcgcagta ctggcagcgg 9540tgcacgggct gtacatcctg cacgaggttg acctgacgac cgcgcacaag gaagcagagt 9600gggaatttga gcccctcgcc tggcgggttt ggctggtggt cttctacttc ggctgcttgt 9660ccttgaccgt ctggctgctc gaggggagtt acggtggatc ggaccaccac gccgcgcgag 9720cccaaagtcc agatgtccgc gcgcggcggt cggagcttga tgacaacatc gcgcagatgg 9780gagctgtcca tggtctggag ctcccgcggc gtcaggtcag gcgggagctc ctgcaggttt 9840acctcgcata gacgggtcag ggcgcgggct agatccaggt gatacctaat ttccaggggc 9900tggttggtgg cggcgtcgat ggcttgcaag aggccgcatc cccgcggcgc gactacggta 9960ccgcgcggcg ggcggtgggc cgcgggggtg tccttggatg atgcatctaa aagcggtgac 10020gcgggcgagc ccccggaggt agggggggct ccggacccgc cgggagaggg ggcaggggca 10080cgtcggcgcc gcgcgcgggc aggagctggt gctgcgcgcg taggttgctg gcgaacgcga 10140cgacgcggcg gttgatctcc tgaatctggc gcctctgcgt gaagacgacg ggcccggtga 10200gcttgagcct gaaagagagt tcgacagaat caatttcggt gtcgttgacg gcggcctggc 10260gcaaaatctc ctgcacgtct cctgagttgt cttgataggc gatctcggcc atgaactgct 10320cgatctcttc ctcctggaga tctccgcgtc cggctcgctc cacggtggcg gcgaggtcgt 10380tggaaatgcg ggccatgagc tgcgagaagg cgttgaggcc tccctcgttc cagacgcggc 10440tgtagaccac gcccccttcg gcatcgcggg cgcgcatgac cacctgcgcg agattgagct 10500ccacgtgccg ggcgaagacg gcgtagtttc gcaggcgctg aaagaggtag ttgagggtgg 10560tggcggtgtg ttctgccacg aagaagtaca taacccagcg tcgcaacgtg gattcgttga 10620tatcccccaa ggcctcaagg cgctccatgg cctcgtagaa gtccacggcg aagttgaaaa 10680actgggagtt gcgcgccgac acggttaact cctcctccag aagacggatg agctcggcga 10740cagtgtcgcg cacctcgcgc tcaaaggcta caggggcctc ttcttcttct tcaatctcct 10800cttccataag ggcctcccct tcttcttctt ctggcggcgg tgggggaggg gggacacggc 10860ggcgacgacg gcgcaccggg aggcggtcga caaagcgctc gatcatctcc ccgcggcgac 10920ggcgcatggt ctcggtgacg gcgcggccgt tctcgcgggg gcgcagttgg aagacgccgc 10980ccgtcatgtc ccggttatgg gttggcgggg ggctgccatg cggcagggat acggcgctaa 11040cgatgcatct caacaattgt tgtgtaggta ctccgccgcc gagggacctg agcgagtccg 11100catcgaccgg atcggaaaac ctctcgagaa aggcgtctaa ccagtcacag tcgcaaggta 11160ggctgagcac cgtggcgggc ggcagcgggc ggcggtcggg gttgtttctg gcggaggtgc 11220tgctgatgat gtaattaaag taggcggtct tgagacggcg gatggtcgac agaagcacca 11280tgtccttggg tccggcctgc tgaatgcgca ggcggtcggc catgccccag gcttcgtttt 11340gacatcggcg caggtctttg tagtagtctt gcatgagcct ttctaccggc acttcttctt 11400ctccttcctc ttgtcctgca tctcttgcat ctatcgctgc ggcggcggcg gagtttggcc 11460gtaggtggcg ccctcttcct cccatgcgtg tgaccccgaa gcccctcatc ggctgaagca 11520gggctaggtc ggcgacaacg cgctcggcta atatggcctg ctgcacctgc gtgagggtag 11580actggaagtc atccatgtcc acaaagcggt ggtatgcgcc cgtgttgatg gtgtaagtgc 11640agttggccat aacggaccag ttaacggtct ggtgacccgg ctgcgagagc tcggtgtacc 11700tgagacgcga gtaagccctc gagtcaaata cgtagtcgtt gcaagtccgc accaggtact 11760ggtatcccac caaaaagtgc ggcggcggct ggcggtagag gggccagcgt agggtggccg 11820gggctccggg ggcgagatct tccaacataa ggcgatgata tccgtagatg tacctggaca 11880tccaggtgat gccggcggcg gtggtggagg cgcgcggaaa gtcgcggacg cggttccaga 11940tgttgcgcag cggcaaaaag tgctccatgg tcgggacgct ctggccggtc aggcgcgcgc 12000aatcgttgac gctctagacc gtgcaaaagg agagcctgta agcgggcact cttccgtggt 12060ctggtggata aattcgcaag ggtatcatgg cggacgaccg gggttcgagc cccgtatccg 12120gccgtccgcc gtgatccatg cggttaccgc ccgcgtgtcg aacccaggtg tgcgacgtca 12180gacaacgggg gagtgctcct tttggcttcc ttccaggcgc ggcggctgct gcgctagctt 12240ttttggccac tggccgcgcg cagcgtaagc ggttaggctg gaaagcgaaa gcattaagtg 12300gctcgctccc tgtagccgga gggttatttt ccaagggttg agtcgcggga cccccggttc 12360gagtctcgga ccggccggac tgcggcgaac gggggtttgc ctccccgtca tgcaagaccc 12420cgcttgcaaa ttcctccgga aacagggacg agcccctttt ttgcttttcc cagatgcatc 12480cggtgctgcg gcagatgcgc ccccctcctc agcagcggca agagcaagag cagcggcaga 12540catgcagggc accctcccct cctcctaccg cgtcaggagg ggcgacatcc gcggttgacg 12600cggcagcaga tggtgattac gaacccccgc ggcgccgggc ccggcactac ctggacttgg 12660aggagggcga gggcctggcg cggctaggag cgccctctcc tgagcggtac ccaagggtgc 12720agctgaagcg tgatacgcgt gaggcgtacg tgccgcggca gaacctgttt cgcgaccgcg 12780agggagagga gcccgaggag atgcgggatc gaaagttcca cgcagggcgc gagctgcggc 12840atggcctgaa tcgcgagcgg ttgctgcgcg aggaggactt tgagcccgac gcgcgaaccg 12900ggattagtcc cgcgcgcgca cacgtggcgg ccgccgacct ggtaaccgca tacgagcaga 12960cggtgaacca ggagattaac tttcaaaaaa gctttaacaa ccacgtgcgt acgcttgtgg 13020cgcgcgagga ggtggctata ggactgatgc atctgtggga ctttgtaagc gcgctggagc 13080aaaacccaaa tagcaagccg ctcatggcgc agctgttcct tatagtgcag cacagcaggg 13140acaacgaggc attcagggat gcgctgctaa acatagtaga gcccgagggc cgctggctgc 13200tcgatttgat aaacatcctg cagagcatag tggtgcagga gcgcagcttg agcctggctg 13260acaaggtggc cgccatcaac tattccatgc ttagcctggg caagttttac gcccgcaaga 13320tataccatac cccttacgtt cccatagaca aggaggtaaa gatcgagggg ttctacatgc 13380gcatggcgct gaaggtgctt accttgagcg acgacctggg cgtttatcgc aacgagcgca 13440tccacaaggc cgtgagcgtg agccggcggc gcgagctcag cgaccgcgag ctgatgcaca 13500gcctgcaaag ggccctggct ggcacgggca gcggcgatag agaggccgag tcctactttg 13560acgcgggcgc tgacctgcgc tgggccccaa gccgacgcgc cctggaggca gctggggccg 13620gacctgggct ggcggtggca cccgcgcgcg ctggcaacgt cggcggcgtg gaggaatatg 13680acgaggacga tgagtacgag ccagaggacg gcgagtacta agcggtgatg tttctgatca 13740gatgatgcaa gacgcaacgg acccggcggt gcgggcggcg ctgcagagcc agccgtccgg 13800ccttaactcc acggacgact ggcgccaggt catggaccgc atcatgtcgc tgactgcgcg 13860caatcctgac gcgttccggc agcagccgca ggccaaccgg ctctccgcaa ttctggaagc 13920ggtggtcccg gcgcgcgcaa accccacgca cgagaaggtg ctggcgatcg taaacgcgct 13980ggccgaaaac agggccatcc ggcccgacga ggccggcctg gtctacgacg cgctgcttca 14040gcgcgtggct cgttacaaca gcggcaacgt gcagaccaac ctggaccggc tggtggggga 14100tgtgcgcgag gccgtggcgc agcgtgagcg cgcgcagcag cagggcaacc tgggctccat 14160ggttgcacta aacgccttcc tgagtacaca gcccgccaac gtgccgcggg gacaggagga 14220ctacaccaac tttgtgagcg cactgcggct aatggtgact gagacaccgc aaagtgaggt 14280gtaccagtct gggccagact attttttcca gaccagtaga caaggcctgc agaccgtaaa 14340cctgagccag gctttcaaaa acttgcaggg gctgtggggg gtgcgggctc ccacaggcga 14400ccgcgcgacc gtgtctagct tgctgacgcc caactcgcgc ctgttgctgc tgctaatagc 14460gcccttcacg gacagtggca gcgtgtcccg ggacacatac ctaggtcact tgctgacact 14520gtaccgcgag gccataggtc aggcgcatgt ggacgagcat actttccagg agattacaag 14580tgtcagccgc gcgctggggc aggaggacac gggcagcctg gaggcaaccc taaactacct 14640gctgaccaac cggcggcaga agatcccctc gttgcacagt ttaaacagcg aggaggagcg 14700cattttgcgc tacgtgcagc agagcgtgag ccttaacctg atgcgcgacg gggtaacgcc 14760cagcgtggcg ctggacatga ccgcgcgcaa catggaaccg ggcatgtatg cctcaaaccg 14820gccgtttatc aaccgcctaa tggactactt gcatcgcgcg gccgccgtga accccgagta 14880tttcaccaat gccatcttga acccgcactg gctaccgccc cctggtttct acaccggggg 14940attcgaggtg cccgagggta acgatggatt cctctgggac gacatagacg acagcgtgtt 15000ttccccgcaa ccgcagaccc tgctagagtt gcaacagcgc gagcaggcag aggcggcgct 15060gcgaaaggaa agcttccgca ggccaagcag cttgtccgat ctaggcgctg cggccccgcg 15120gtcagatgct agtagcccat ttccaagctt gatagggtct cttaccagca ctcgcaccac 15180ccgcccgcgc ctgctgggcg aggaggagta cctaaacaac tcgctgctgc agccgcagcg 15240cgaaaaaaac ctgcctccgg catttcccaa caacgggata gagagcctag tggacaagat 15300gagtagatgg aagacgtacg cgcaggagca cagggacgtg ccaggcccgc gcccgcccac 15360ccgtcgtcaa aggcacgacc gtcagcgggg tctggtgtgg gaggacgatg actcggcaga 15420cgacagcagc gtcctggatt tgggagggag tggcaacccg tttgcgcacc ttcgccccag 15480gctggggaga atgttttaaa aaaaaaaaag catgatgcaa aataaaaaac tcaccaaggc 15540catggcaccg agcgttggtt ttcttgtatt ccccttagta tgcggcgcgc ggcgatgtat 15600gaggaaggtc ctcctccctc ctacgagagt gtggtgagcg cggcgccagt ggcggcggcg 15660ctgggttctc ccttcgatgc tcccctggac ccgccgtttg tgcctccgcg gtacctgcgg 15720cctaccgggg ggagaaacag catccgttac tctgagttgg cacccctatt cgacaccacc 15780cgtgtgtacc tggtggacaa caagtcaacg gatgtggcat ccctgaacta ccagaacgac 15840cacagcaact ttctgaccac ggtcattcaa aacaatgact acagcccggg ggaggcaagc 15900acacagacca tcaatcttga cgaccggtcg cactggggcg gcgacctgaa aaccatcctg 15960cataccaaca tgccaaatgt gaacgagttc atgtttacca ataagtttaa ggcgcgggtg 16020atggtgtcgc gcttgcctac taaggacaat caggtggagc tgaaatacga gtgggtggag 16080ttcacgctgc ccgagggcaa ctactccgag accatgacca tagaccttat gaacaacgcg 16140atcgtggagc actacttgaa agtgggcaga cagaacgggg ttctggaaag cgacatcggg 16200gtaaagtttg acacccgcaa cttcagactg gggtttgacc ccgtcactgg tcttgtcatg 16260cctggggtat atacaaacga agccttccat ccagacatca ttttgctgcc aggatgcggg 16320gtggacttca cccacagccg cctgagcaac ttgttgggca tccgcaagcg gcaacccttc 16380caggagggct ttaggatcac ctacgatgat ctggagggtg gtaacattcc cgcactgttg 16440gatgtggacg cctaccaggc gagcttgaaa gatgacaccg aacagggcgg gggtggcgca 16500ggcggcagca acagcagtgg cagcggcgcg gaagagaact ccaacgcggc agccgcggca 16560atgcagccgg tggaggacat gaacgatcat gccattcgcg gcgacacctt tgccacacgg 16620gctgaggaga agcgcgctga ggccgaagca gcggccgaag ctgccgcccc cgctgcgcaa 16680cccgaggtcg agaagcctca gaagaaaccg gtgatcaaac ccctgacaga ggacagcaag 16740aaacgcagtt acaacctaat aagcaatgac agcaccttca cccagtaccg cagctggtac 16800cttgcataca actacggcga ccctcagacc ggaatccgct catggaccct gctttgcact 16860cctgacgtaa cctgcggctc ggagcaggtc tactggtcgt tgccagacat gatgcaagac 16920cccgtgacct tccgctccac gcgccagatc agcaactttc cggtggtggg cgccgagctg 16980ttgcccgtgc actccaagag cttctacaac gaccaggccg tctactccca actcatccgc 17040cagtttacct ctctgaccca cgtgttcaat cgctttcccg agaaccagat tttggcgcgc 17100ccgccagccc ccaccatcac caccgtcagt gaaaacgttc ctgctctcac agatcacggg 17160acgctaccgc tgcgcaacag catcggagga gtccagcgag tgaccattac tgacgccaga 17220cgccgcacct gcccctacgt ttacaaggcc ctgggcatag tctcgccgcg cgtcctatcg 17280agccgcactt tttgagcaag catgtccatc cttatatcgc ccagcaataa cacaggctgg 17340ggcctgcgct tcccaagcaa gatgtttggc ggggccaaga agcgctccga ccaacaccca 17400gtgcgcgtgc gcgggcacta ccgcgcgccc tggggcgcgc acaaacgcgg ccgcactggg 17460cgcaccaccg tcgatgacgc catcgacgcg gtggtggagg aggcgcgcaa ctacacgccc 17520acgccgccac cagtgtccac agtggacgcg gccattcaga ccgtggtgcg cggagcccgg 17580cgctatgcta aaatgaagag acggcggagg cgcgtagcac gtcgccaccg ccgccgaccc 17640ggcactgccg cccaacgcgc ggcggcggcc ctgcttaacc gcgcacgtcg caccggccga 17700cgggcggcca tgcgggccgc tcgaaggctg gccgcgggta ttgtcactgt gccccccagg 17760tccaggcgac gagcggccgc cgcagcagcc gcggccatta gtgctatgac tcagggtcgc 17820aggggcaacg tgtattgggt gcgcgactcg gttagcggcc tgcgcgtgcc cgtgcgcacc 17880cgccccccgc gcaactagat tgcaagaaaa aactacttag actcgtactg ttgtatgtat 17940ccagcggcgg cggcgcgcaa cgaagctatg tccaagcgca aaatcaaaga agagatgctc 18000caggtcatcg cgccggagat ctatggcccc ccgaagaagg aagagcagga ttacaagccc 18060cgaaagctaa agcgggtcaa aaagaaaaag aaagatgatg atgatgaact tgacgacgag 18120gtggaactgc tgcacgctac cgcgcccagg cgacgggtac agtggaaagg tcgacgcgta 18180aaacgtgttt tgcgacccgg caccaccgta gtctttacgc ccggtgagcg ctccacccgc 18240acctacaagc gcgtgtatga tgaggtgtac ggcgacgagg acctgcttga gcaggccaac 18300gagcgcctcg gggagtttgc ctacggaaag cggcataagg acatgctggc gttgccgctg 18360gacgagggca acccaacacc tagcctaaag cccgtaacac tgcagcaggt gctgcccgcg 18420cttgcaccgt ccgaagaaaa gcgcggccta aagcgcgagt ctggtgactt ggcacccacc 18480gtgcagctga tggtacccaa gcgccagcga ctggaagatg tcttggaaaa aatgaccgtg 18540gaacctgggc tggagcccga ggtccgcgtg cggccaatca agcaggtggc gccgggactg 18600ggcgtgcaga ccgtggacgt tcagataccc actaccagta gcaccagtat tgccaccgcc 18660acagagggca tggagacaca aacgtccccg gttgcctcag cggtggcgga tgccgcggtg 18720caggcggtcg ctgcggccgc gtccaagacc tctacggagg tgcaaacgga cccgtggatg 18780tttcgcgttt cagccccccg gcgcccgcgc ggttcgagga agtacggcgc cgccagcgcg 18840ctactgcccg aatatgccct acatccttcc attgcgccta cccccggcta tcgtggctac 18900acctaccgcc ccagaagacg agcaactacc cgacgccgaa ccaccactgg aacccgccgc 18960cgccgtcgcc gtcgccagcc cgtgctggcc ccgatttccg tgcgcagggt ggctcgcgaa 19020ggaggcagga ccctggtgct gccaacagcg cgctaccacc ccagcatcgt ttaaaagccg 19080gtctttgtgg ttcttgcaga tatggccctc acctgccgcc tccgtttccc ggtgccggga 19140ttccgaggaa gaatgcaccg taggaggggc atggccggcc acggcctgac gggcggcatg 19200cgtcgtgcgc accaccggcg gcggcgcgcg tcgcaccgtc gcatgcgcgg cggtatcctg 19260cccctcctta ttccactgat cgccgcggcg attggcgccg tgcccggaat tgcatccgtg 19320gccttgcagg cgcagagaca ctgattaaaa acaagttgca tgtggaaaaa tcaaaataaa 19380aagtctggac tctcacgctc gcttggtcct gtaactattt tgtagaatgg aagacatcaa 19440ctttgcgtct ctggccccgc gacacggctc gcgcccgttc atgggaaact ggcaagatat 19500cggcaccagc aatatgagcg gtggcgcctt cagctggggc tcgctgtgga gcggcattaa 19560aaatttcggt tccaccgtta agaactatgg cagcaaggcc tggaacagca gcacaggcca 19620gatgctgagg gataagttga aagagcaaaa tttccaacaa aaggtggtag atggcctggc 19680ctctggcatt agcggggtgg tggacctggc caaccaggca gtgcaaaata agattaacag 19740taagcttgat ccccgccctc ccgtagagga gcctccaccg gccgtggaga cagtgtctcc 19800agaggggcgt ggcgaaaagc gtccgcgccc cgacagggaa gaaactctgg tgacgcaaat 19860agacgagcct ccctcgtacg aggaggcact aaagcaaggc ctgcccacca cccgtcccat 19920cgcgcccatg gctaccggag tgctgggcca gcacacaccc gtaacgctgg acctgcctcc 19980ccccgccgac acccagcaga aacctgtgct gccaggcccg accgccgttg ttgtaacccg 20040tcctagccgc gcgtccctgc gccgcgccgc cagcggtccg cgatcgttgc ggcccgtagc 20100cagtggcaac tggcaaagca cactgaacag catcgtgggt ctgggggtgc aatccctgaa 20160gcgccgacga tgcttctgaa tagctaacgt gtcgtatgtg tgtcatgtat gcgtccatgt 20220cgccgccaga ggagctgctg agccgccgcg cgcccgcttt ccaagatggc taccccttcg 20280atgatgccgc agtggtctta catgcacatc tcgggccagg acgcctcgga gtacctgagc 20340cccgggctgg tgcagtttgc ccgcgccacc gagacgtact tcagcctgaa taacaagttt 20400agaaacccca cggtggcgcc tacgcacgac gtgaccacag accggtccca gcgtttgacg 20460ctgcggttca tccctgtgga ccgtgaggat actgcgtact cgtacaaggc gcggttcacc 20520ctagctgtgg gtgataaccg tgtgctggac atggcttcca cgtactttga catccgcggc 20580gtgctggaca ggggccctac ttttaagccc tactctggca ctgcctacaa cgccctggct 20640cccaagggtg ccccaaatcc ttgcgaatgg gatgaagctg ctactgctct tgaaataaac 20700ctagaagaag aggacgatga caacgaagac gaagtagacg agcaagctga gcagcaaaaa 20760actcacgtat ttgggcaggc gccttattct ggtataaata ttacaaagga gggtattcaa 20820ataggtgtcg aaggtcaaac acctaaatat gccgataaaa catttcaacc tgaacctcaa 20880ataggagaat ctcagtggta cgaaactgaa attaatcatg cagctgggag agtccttaaa 20940aagactaccc caatgaaacc atgttacggt tcatatgcaa aacccacaaa tgaaaatgga 21000gggcaaggca ttcttgtaaa gcaacaaaat ggaaagctag aaagtcaagt ggaaatgcaa 21060tttttctcaa ctactgaggc gaccgcaggc aatggtgata acttgactcc taaagtggta 21120ttgtacagtg aagatgtaga tatagaaacc ccagacactc atatttctta catgcccact 21180attaaggaag gtaactcacg agaactaatg ggccaacaat ctatgcccaa caggcctaat 21240tacattgctt ttagggacaa ttttattggt ctaatgtatt acaacagcac gggtaatatg 21300ggtgttctgg cgggccaagc atcgcagttg aatgctgttg tagatttgca agacagaaac 21360acagagcttt cataccagct tttgcttgat tccattggtg atagaaccag gtacttttct 21420atgtggaatc aggctgttga cagctatgat ccagatgtta gaattattga aaatcatgga 21480actgaagatg aacttccaaa ttactgcttt ccactgggag gtgtgattaa tacagagact 21540cttaccaagg taaaacctaa aacaggtcag gaaaatggat gggaaaaaga tgctacagaa 21600ttttcagata aaaatgaaat aagagttgga aataattttg ccatggaaat caatctaaat 21660gccaacctgt ggagaaattt cctgtactcc aacatagcgc tgtatttgcc cgacaagcta 21720aagtacagtc cttccaacgt aaaaatttct gataacccaa acacctacga ctacatgaac 21780aagcgagtgg tggctcccgg gttagtggac tgctacatta accttggagc acgctggtcc 21840cttgactata tggacaacgt caacccattt aaccaccacc gcaatgctgg cctgcgctac 21900cgctcaatgt tgctgggcaa tggtcgctat gtgcccttcc acatccaggt gcctcagaag 21960ttctttgcca ttaaaaacct ccttctcctg ccgggctcat acacctacga gtggaacttc 22020aggaaggatg ttaacatggt tctgcagagc tccctaggaa atgacctaag ggttgacgga 22080gccagcatta agtttgatag catttgcctt tacgccacct tcttccccat ggcccacaac 22140accgcctcca cgcttgaggc catgcttaga aacgacacca acgaccagtc ctttaacgac 22200tatctctccg ccgccaacat gctctaccct atacccgcca acgctaccaa cgtgcccata 22260tccatcccct cccgcaactg ggcggctttc cgcggctggg ccttcacgcg ccttaagact 22320aaggaaaccc catcactggg ctcgggctac gacccttatt acacctactc tggctctata 22380ccctacctag atggaacctt ttacctcaac cacaccttta agaaggtggc cattaccttt 22440gactcttctg tcagctggcc tggcaatgac cgcctgctta cccccaacga gtttgaaatt 22500aagcgctcag ttgacgggga gggttacaac gttgcccagt gtaacatgac caaagactgg 22560ttcctggtac aaatgctagc taactacaac attggctacc agggcttcta tatcccagag 22620agctacaagg accgcatgta ctccttcttt agaaacttcc agcccatgag ccgtcaggtg 22680gtggatgata ctaaatacaa ggactaccaa caggtgggca tcctacacca acacaacaac 22740tctggatttg ttggctacct tgcccccacc atgcgcgaag gacaggccta ccctgctaac 22800ttcccctatc cgcttatagg caagaccgca gttgacagca ttacccagaa aaagtttctt 22860tgcgatcgca ccctttggcg catcccattc tccagtaact ttatgtccat gggcgcactc 22920acagacctgg gccaaaacct tctctacgcc aactccgccc acgcgctaga catgactttt 22980gaggtggatc ccatggacga gcccaccctt ctttatgttt tgtttgaagt ctttgacgtg 23040gtccgtgtgc accggccgca ccgcggcgtc atcgaaaccg tgtacctgcg cacgcccttc 23100tcggccggca acgccacaac ataaagaagc aagcaacatc aacaacagct gccgccatgg 23160gctccagtga gcaggaactg aaagccattg tcaaagatct tggttgtggg ccatattttt 23220tgggcaccta tgacaagcgc tttccaggct ttgtttctcc acacaagctc gcctgcgcca 23280tagtcaatac ggccggtcgc gagactgggg gcgtacactg gatggccttt gcctggaacc 23340cgcactcaaa aacatgctac ctctttgagc cctttggctt ttctgaccag cgactcaagc 23400aggtttacca gtttgagtac gagtcactcc tgcgccgtag cgccattgct tcttcccccg 23460accgctgtat aacgctggaa aagtccaccc aaagcgtaca ggggcccaac tcggccgcct 23520gtggactatt ctgctgcatg tttctccacg cctttgccaa ctggccccaa actcccatgg 23580atcacaaccc caccatgaac cttattaccg gggtacccaa ctccatgctc aacagtcccc 23640aggtacagcc caccctgcgt cgcaaccagg aacagctcta cagcttcctg gagcgccact 23700cgccctactt ccgcagccac agtgcgcaga ttaggagcgc cacttctttt tgtcacttga 23760aaaacatgta aaaataatgt actagagaca ctttcaataa aggcaaatgc ttttatttgt 23820acactctcgg gtgattattt acccccaccc ttgccgtctg cgccgtttaa aaatcaaagg 23880ggttctgccg cgcatcgcta tgcgccactg gcagggacac gttgcgatac tggtgtttag 23940tgctccactt aaactcaggc acaaccatcc gcggcagctc ggtgaagttt tcactccaca 24000ggctgcgcac catcaccaac gcgtttagca ggtcgggcgc cgatatcttg aagtcgcagt 24060tggggcctcc gccctgcgcg cgcgagttgc gatacacagg gttgcagcac tggaacacta 24120tcagcgccgg gtggtgcacg ctggccagca cgctcttgtc ggagatcaga tccgcgtcca 24180ggtcctccgc gttgctcagg gcgaacggag tcaactttgg tagctgcctt cccaaaaagg 24240gcgcgtgccc aggctttgag ttgcactcgc accgtagtgg catcaaaagg tgaccgtgcc 24300cggtctgggc gttaggatac agcgcctgca taaaagcctt gatctgctta aaagccacct 24360gagcctttgc gccttcagag aagaacatgc cgcaagactt gccggaaaac tgattggccg 24420gacaggccgc gtcgtgcacg cagcaccttg cgtcggtgtt ggagatctgc accacatttc 24480ggccccaccg gttcttcacg atcttggcct tgctagactg ctccttcagc gcgcgctgcc 24540cgttttcgct cgtcacatcc atttcaatca cgtgctcctt atttatcata atgcttccgt 24600gtagacactt aagctcgcct tcgatctcag cgcagcggtg cagccacaac gcgcagcccg 24660tgggctcgtg atgcttgtag gtcacctctg caaacgactg caggtacgcc tgcaggaatc 24720gccccatcat cgtcacaaag gtcttgttgc tggtgaaggt cagctgcaac ccgcggtgct 24780cctcgttcag ccaggtcttg catacggccg ccagagcttc cacttggtca ggcagtagtt 24840tgaagttcgc ctttagatcg ttatccacgt ggtacttgtc catcagcgcg cgcgcagcct 24900ccatgccctt ctcccacgca gacacgatcg gcacactcag cgggttcatc accgtaattt 24960cactttccgc ttcgctgggc tcttcctctt cctcttgcgt ccgcatacca cgcgccactg 25020ggtcgtcttc attcagccgc cgcactgtgc gcttacctcc tttgccatgc ttgattagca 25080ccggtgggtt gctgaaaccc accatttgta gcgccacatc ttctctttct tcctcgctgt 25140ccacgattac ctctggtgat ggcgggcgct cgggcttggg agaagggcgc ttctttttct 25200tcttgggcgc aatggccaaa tccgccgccg aggtcgatgg ccgcgggctg ggtgtgcgcg 25260gcaccagcgc gtcttgtgat gagtcttcct cgtcctcgga ctcgatacgc cgcctcatcc 25320gcttttttgg gggcgcccgg ggaggcggcg gcgacgggga cggggacgac acgtcctcca 25380tggttggggg acgtcgcgcc gcaccgcgtc cgcgctcggg ggtggtttcg cgctgctcct 25440cttcccgact ggccatttcc ttctcctata ggcagaaaaa gatcatggag tcagtcgaga 25500agaaggacag cctaaccgcc ccctctgagt tcgccaccac cgcctccacc gatgccgcca 25560acgcgcctac caccttcccc gtcgaggcac ccccgcttga ggaggaggaa gtgattatcg 25620agcaggaccc aggttttgta agcgaagacg acgaggaccg ctcagtacca acagaggata 25680aaaagcaaga ccaggacaac gcagaggcaa acgaggaaca agtcgggcgg ggggacgaaa 25740ggcatggcga ctacctagat gtgggagacg acgtgctgtt gaagcatctg cagcgccagt 25800gcgccattat ctgcgacgcg ttgcaagagc gcagcgatgt gcccctcgcc atagcggatg 25860tcagccttgc ctacgaacgc cacctattct caccgcgcgt accccccaaa cgccaagaaa 25920acggcacatg cgagcccaac ccgcgcctca acttctaccc cgtatttgcc gtgccagagg 25980tgcttgccac ctatcacatc tttttccaaa actgcaagat acccctatcc tgccgtgcca 26040accgcagccg agcggacaag cagctggcct tgcggcaggg cgctgtcata cctgatatcg 26100cctcgctcaa cgaagtgcca aaaatctttg agggtcttgg acgcgacgag aagcgcgcgg 26160caaacgctct gcaacaggaa aacagcgaaa atgaaagtca ctctggagtg ttggtggaac 26220tcgagggtga caacgcgcgc ctagccgtac taaaacgcag catcgaggtc acccactttg 26280cctacccggc acttaaccta ccccccaagg tcatgagcac agtcatgagt gagctgatcg 26340tgcgccgtgc gcagcccctg gagagggatg caaatttgca agaacaaaca gaggagggcc 26400tacccgcagt tggcgacgag cagctagcgc gctggcttca aacgcgcgag cctgccgact 26460tggaggagcg acgcaaacta atgatggccg cagtgctcgt taccgtggag cttgagtgca 26520tgcagcggtt ctttgctgac ccggagatgc agcgcaagct agaggaaaca ttgcactaca 26580cctttcgaca gggctacgta cgccaggcct gcaagatctc caacgtggag ctctgcaacc 26640tggtctccta ccttggaatt ttgcacgaaa accgccttgg gcaaaacgtg cttcattcca 26700cgctcaaggg cgaggcgcgc cgcgactacg tccgcgactg cgtttactta tttctatgct 26760acacctggca gacggccatg ggcgtttggc agcagtgctt ggaggagtgc aacctcaagg 26820agctgcagaa actgctaaag caaaacttga aggacctatg gacggccttc aacgagcgct 26880ccgtggccgc gcacctggcg gacatcattt tccccgaacg cctgcttaaa accctgcaac 26940agggtctgcc agacttcacc agtcaaagca tgttgcagaa ctttaggaac tttatcctag 27000agcgctcagg aatcttgccc gccacctgct gtgcacttcc tagcgacttt gtgcccatta 27060agtaccgcga atgccctccg ccgctttggg gccactgcta ccttctgcag ctagccaact 27120accttgccta ccactctgac ataatggaag acgtgagcgg tgacggtcta ctggagtgtc 27180actgtcgctg caacctatgc accccgcacc gctccctggt ttgcaattcg cagctgctta 27240acgaaagtca aattatcggt acctttgagc tgcagggtcc ctcgcctgac gaaaagtccg 27300cggctccggg gttgaaactc actccggggc tgtggacgtc ggcttacctt cgcaaatttg 27360tacctgagga ctaccacgcc cacgagatta ggttctacga agaccaatcc cgcccgccaa 27420atgcggagct taccgcctgc gtcattaccc agggccacat tcttggccaa ttgcaagcca 27480tcaacaaagc ccgccaagag tttctgctac gaaagggacg gggggtttac ttggaccccc 27540agtccggcga ggagctcaac ccaatccccc cgccgccgca gccctatcag cagcagccgc 27600gggcccttgc ttcccaggat ggcacccaaa aagaagctgc agctgccgcc gccacccacg 27660gacgaggagg aatactggga cagtcaggca gaggaggttt tggacgagga ggaggaggac 27720atgatggaag actgggagag cctagacgag gaagcttccg aggtcgaaga ggtgtcagac 27780gaaacaccgt caccctcggt cgcattcccc tcgccggcgc cccagaaatc ggcaaccggt 27840tccagcatgg ctacaacctc cgctcctcag gcgccgccgg cactgcccgt tcgccgaccc 27900aaccgtagat gggacaccac tggaaccagg gccggtaagt ccaagcagcc gccgccgtta 27960gcccaagagc aacaacagcg ccaaggctac cgctcatggc gcgggcacaa gaacgccata 28020gttgcttgct tgcaagactg tgggggcaac atctccttcg cccgccgctt tcttctctac 28080catcacggcg tggccttccc ccgtaacatc ctgcattact accgtcatct ctacagccca 28140tactgcaccg gcggcagcgg cagcggcagc aacagcagcg gccacacaga agcaaaggcg 28200accggatagc aagactctga caaagcccaa gaaatccaca gcggcggcag cagcaggagg 28260aggagcgctg cgtctggcgc ccaacgaacc cgtatcgacc cgcgagctta gaaacaggat 28320ttttcccact ctgtatgcta tatttcaaca gagcaggggc caagaacaag agctgaaaat 28380aaaaaacagg tctctgcgat ccctcacccg cagctgcctg tatcacaaaa gcgaagatca 28440gcttcggcgc acgctggaag acgcggaggc tctcttcagt aaatactgcg cgctgactct 28500taaggactag tttcgcgccc tttctcaaat ttaagcgcga aaactacgtc atctccagcg 28560gccacacccg gcgccagcac ctgtcgtcag cgccattatg agcaaggaaa ttcccacgcc 28620ctacatgtgg agttaccagc cacaaatggg acttgcggct ggagctgccc aagactactc 28680aacccgaata aactacatga gcgcgggacc ccacatgata tcccgggtca acggaatccg 28740cgcccaccga aaccgaattc tcttggaaca ggcggctatt accaccacac ctcgtaataa 28800ccttaatccc cgtagttggc ccgctgccct ggtgtaccag gaaagtcccg ctcccaccac 28860tgtggtactt cccagagacg cccaggccga agttcagatg actaactcag gggcgcagct 28920tgcgggcggc tttcgtcaca gggtgcggtc gcccgggcag ggtataactc acctgacaat 28980cagagggcga ggtattcagc tcaacgacga gtcggtgagc tcctcgcttg gtctccgtcc 29040ggacgggaca tttcagatcg gcggcgccgg ccgtccttca ttcacgcctc gtcaggcaat 29100cctaactctg cagacctcgt cctctgagcc gcgctctgga ggcattggaa ctctgcaatt 29160tattgaggag tttgtgccat cggtctactt taaccccttc tcgggacctc ccggccacta 29220tccggatcaa tttattccta actttgacgc ggtaaaggac tcggcggacg gctacgactg 29280aatgttaagt ggagaggcag agcaactgcg cctgaaacac ctggtccact gtcgccgcca 29340caagtgcttt gcccgcgact ccggtgagtt ttgctacttt gaattgcccg aggatcatat 29400cgagggcccg gcgcacggcg tccggcttac cgcccaggga gagcttgccc gtagcctgat 29460tcgggagttt acccagcgcc ccctgctagt tgagcgggac aggggaccct gtgttctcac 29520tgtgatttgc aactgtccta accttggatt acatcaagat ctttgttgcc atctctgtgc 29580tgagtataat aaatacagaa attaaaatat actggggctc ctatcgccat cctgtaaacg 29640ccaccgtctt cacccgccca agcaaaccaa ggcgaacctt acctggtact tttaacatct 29700ctccctctgt gatttacaac agtttcaacc cagacggagt gagtctacga gagaacctct 29760ccgagctcag ctactccatc agaaaaaaca ccaccctcct tacctgccgg gaacgtacga 29820gtgcgtcacc ggccgctgca ccacacctac cgcctgaccg taaaccagac tttttccgga 29880cagacctcaa taactctgtt taccagaaca ggaggtgagc ttagaaaacc cttagggtat 29940taggccaaag gcgcagctac tgtggggttt atgaacaatt caagcaactc tacgggctat 30000tctaattcag gtttctctag aaatggacgg aattattaca gagcagcgcc tgctagaaag 30060acgcagggca gcggccgagc aacagcgcat gaatcaagag ctccaagaca tggttaactt 30120gcaccagtgc aaaaggggta tcttttgtct ggtaaagcag gccaaagtca cctacgacag 30180taataccacc ggacaccgcc ttagctacaa gttgccaacc aagcgtcaga aattggtggt 30240catggtggga gaaaagccca ttaccataac tcagcactcg gtagaaaccg aaggctgcat 30300tcactcacct tgtcaaggac ctgaggatct ctgcaccctt attaagaccc tgtgcggtct 30360caaagatctt attcccttta actaataaaa aaaaataata aagcatcact tacttaaaat 30420cagttagcaa atttctgtcc agtttattca gcagcacctc cttgccctcc tcccagctct 30480ggtattgcag cttcctcctg gctgcaaact ttctccacaa tctaaatgga atgtcagttt 30540cctcctgttc ctgtccatcc gcacccacta tcttcatgtt gttgcagatg aagcgcgcaa 30600gaccgtctga agataccttc aaccccgtgt atccatatga cacggaaacc ggtcctccaa 30660ctgtgccttt tcttactcct ccctttgtat cccccaatgg gtttcaagag agtccccctg 30720gggtactctc tttgcgccta tccgaacctc tagttacctc caatggcatg cttgcgctca 30780aaatgggcaa cggcctctct ctggacgagg ccggcaacct tacctcccaa aatgtaacca 30840ctgtgagccc acctctcaaa aaaaccaagt caaacataaa cctggaaata tctgcacccc 30900tcacagttac ctcagaagcc ctaactgtgg ctgccgccgc acctctaatg gtcgcgggca 30960acacactcac catgcaatca caggccccgc taaccgtgca cgactccaaa cttagcattg 31020ccacccaagg acccctcaca gtgtcagaag gaaagctagc cctgcaaaca tcaggccccc 31080tcaccaccac cgatagcagt acccttacta tcactgcctc accccctcta actactgcca 31140ctggtagctt gggcattgac ttgaaagagc ccatttatac acaaaatgga aaactaggac 31200taaagtacgg ggctcctttg catgtaacag acgacctaaa cactttgacc gtagcaactg 31260gtccaggtgt gactattaat aatacttcct tgcaaactaa agttactgga gccttgggtt 31320ttgattcaca aggcaatatg caacttaatg tagcaggagg actaaggatt gattctcaaa 31380acagacgcct tatacttgat gttagttatc cgtttgatgc tcaaaaccaa ctaaatctaa 31440gactaggaca gggccctctt tttataaact cagcccacaa cttggatatt aactacaaca 31500aaggccttta cttgtttaca gcttcaaaca attccaaaaa gcttgaggtt aacctaagca 31560ctgccaaggg gttgatgttt gacgctacag ccatagccat taatgcagga gatgggcttg 31620aatttggttc acctaatgca ccaaacacaa atcccctcaa aacaaaaatt ggccatggcc 31680tagaatttga ttcaaacaag gctatggttc ctaaactagg aactggcctt agttttgaca 31740gcacaggtgc cattacagta ggaaacaaaa ataatgataa gctaactttg tggaccacac 31800cagctccatc tcctaactgt agactaaatg cagagaaaga tgctaaactc actttggtct 31860taacaaaatg tggcagtcaa atacttgcta cagtttcagt tttggctgtt aaaggcagtt 31920tggctccaat atctggaaca gttcaaagtg ctcatcttat tataagattt gacgaaaatg 31980gagtgctact aaacaattcc ttcctggacc cagaatattg gaactttaga aatggagatc 32040ttactgaagg cacagcctat acaaacgctg ttggatttat gcctaaccta tcagcttatc 32100caaaatctca cggtaaaact gccaaaagta acattgtcag tcaagtttac ttaaacggag 32160acaaaactaa acctgtaaca ctaaccatta cactaaacgg tacacaggaa acaggagaca 32220caactccaag tgcatactct atgtcatttt catgggactg gtctggccac aactacatta 32280atgaaatatt tgccacatcc tcttacactt tttcatacat tgcccaagaa taaagaatcg 32340tttgtgttat gtttcaacgt gtttattttt caattgcaga aaatttcgaa tcatttttca 32400ttcagtagta tagccccacc accacatagc ttatacagat caccgtacct taatcaaact 32460cacagaaccc tagtattcaa cctgccacct ccctcccaac acacagagta cacagtcctt 32520tctccccggc tggccttaaa aagcatcata tcatgggtaa cagacatatt cttaggtgtt 32580atattccaca cggtttcctg tcgagccaaa cgctcatcag tgatattaat aaactccccg 32640ggcagctcac ttaagttcat gtcgctgtcc agctgctgag ccacaggctg ctgtccaact 32700tgcggttgct taacgggcgg cgaaggagaa gtccacgcct acatgggggt agagtcataa 32760tcgtgcatca ggatagggcg gtggtgctgc agcagcgcgc gaataaactg ctgccgccgc 32820cgctccgtcc tgcaggaata caacatggca gtggtctcct cagcgatgat tcgcaccgcc 32880cgcagcataa ggcgccttgt cctccgggca cagcagcgca ccctgatctc acttaaatca 32940gcacagtaac tgcagcacag caccacaata ttgttcaaaa tcccacagtg caaggcgctg 33000tatccaaagc tcatggcggg gaccacagaa cccacgtggc catcatacca caagcgcagg 33060tagattaagt ggcgacccct cataaacacg ctggacataa acattacctc ttttggcatg 33120ttgtaattca ccacctcccg gtaccatata aacctctgat taaacatggc gccatccacc 33180accatcctaa accagctggc caaaacctgc ccgccggcta tacactgcag ggaaccggga 33240ctggaacaat gacagtggag agcccaggac tcgtaaccat ggatcatcat gctcgtcatg 33300atatcaatgt tggcacaaca caggcacacg tgcatacact tcctcaggat tacaagctcc 33360tcccgcgtta gaaccatatc ccagggaaca acccattcct gaatcagcgt aaatcccaca 33420ctgcagggaa gacctcgcac gtaactcacg ttgtgcattg tcaaagtgtt acattcgggc 33480agcagcggat gatcctccag tatggtagcg cgggtttctg tctcaaaagg aggtagacga 33540tccctactgt acggagtgcg ccgagacaac cgagatcgtg ttggtcgtag tgtcatgcca 33600aatggaacgc cggacgtagt catatttcct gaagcaaaac caggtgcggg cgtgacaaac 33660agatctgcgt ctccggtctc gccgcttaga tcgctctgtg tagtagttgt agtatatcca 33720ctctctcaaa gcatccaggc gccccctggc ttcgggttct atgtaaactc cttcatgcgc 33780cgctgccctg ataacatcca ccaccgcaga ataagccaca cccagccaac ctacacattc 33840gttctgcgag tcacacacgg gaggagcggg aagagctgga agaaccatgt tttttttttt 33900attccaaaag attatccaaa acctcaaaat gaagatctat taagtgaacg cgctcccctc 33960cggtggcgtg gtcaaactct acagccaaag aacagataat ggcatttgta agatgttgca 34020caatggcttc caaaaggcaa acggccctca cgtccaagtg gacgtaaagg ctaaaccctt 34080cagggtgaat ctcctctata aacattccag caccttcaac catgcccaaa taattctcat 34140ctcgccacct tctcaatata tctctaagca aatcccgaat attaagtccg gccattgtaa 34200aaatctgctc cagagcgccc tccaccttca gcctcaagca gcgaatcatg attgcaaaaa 34260ttcaggttcc tcacagacct gtataagatt caaaagcgga acattaacaa aaataccgcg 34320atcccgtagg tcccttcgca gggccagctg aacataatcg tgcaggtctg cacggaccag 34380cgcggccact tccccgccag gaaccttgac aaaagaaccc acactgatta tgacacgcat 34440actcggagct atgctaacca gcgtagcccc gatgtaagct ttgttgcatg ggcggcgata 34500taaaatgcaa ggtgctgctc aaaaaatcag gcaaagcctc gcgcaaaaaa gaaagcacat 34560cgtagtcatg ctcatgcaga taaaggcagg taagctccgg aaccaccaca gaaaaagaca 34620ccatttttct ctcaaacatg tctgcgggtt tctgcataaa cacaaaataa aataacaaaa 34680aaacatttaa acattagaag cctgtcttac aacaggaaaa acaaccctta taagcataag 34740acggactacg gccatgccgg cgtgaccgta aaaaaactgg tcaccgtgat taaaaagcac 34800caccgacagc tcctcggtca tgtccggagt cataatgtaa gactcggtaa acacatcagg 34860ttgattcaca tcggtcagtg ctaaaaagcg accgaaatag cccgggggaa tacatacccg 34920caggcgtaga gacaacatta cagcccccat aggaggtata acaaaattaa taggagagaa 34980aaacacataa acacctgaaa aaccctcctg cctaggcaaa atagcaccct cccgctccag 35040aacaacatac agcgcttcca cagcggcagc cataacagtc agccttacca gtaaaaaaga 35100aaacctatta aaaaaacacc actcgacacg gcaccagctc aatcagtcac agtgtaaaaa 35160agggccaagt gcagagcgag tatatatagg actaaaaaat gacgtaacgg ttaaagtcca 35220caaaaaacac ccagaaaacc gcacgcgaac ctacgcccag aaacgaaagc caaaaaaccc 35280acaacttcct caaatcgtca cttccgtttt cccacgttac gtcacttccc attttaagaa 35340aactacaatt cccaacacat acaagttact ccgccctaaa acctacgtca cccgccccgt 35400tcccacgccc cgcgccacgt cacaaactcc accccctcat tatcatattg gcttcaatcc 35460aaaataaggt atattattga tgatgttaat taatttaaat ccgcatgcga tatcgagctc 35520tcccgggaat tcggatctgc gacgcgaggc tggatggcct tccccattat gattcttctc 35580gcttccggcg gcatcgggat gcccgcgttg caggccatgc tgtccaggca ggtagatgac 35640gaccatcagg gacagcttca cggccagcaa aaggccagga accgtaaaaa ggccgcgttg 35700ctggcgtttt tccataggct ccgcccccct gacgagcatc acaaaaatcg acgctcaagt 35760cagaggtggc gaaacccgac aggactataa agataccagg cgtttccccc tggaagctcc 35820ctcgtgcgct ctcctgttcc gaccctgccg cttaccggat acctgtccgc ctttctccct 35880tcgggaagcg tggcgctttc tcaatgctca cgctgtaggt atctcagttc ggtgtaggtc 35940gttcgctcca agctgggctg tgtgcacgaa ccccccgttc agcccgaccg ctgcgcctta 36000tccggtaact atcgtcttga gtccaacccg gtaagacacg acttatcgcc actggcagca 36060gccactggta acaggattag cagagcgagg tatgtaggcg gtgctacaga gttcttgaag 36120tggtggccta actacggcta cactagaagg acagtatttg gtatctgcgc tctgctgaag 36180ccagttacct tcggaaaaag agttggtagc tcttgatccg gcaaacaaac caccgctggt 36240agcggtggtt tttttgtttg caagcagcag attacgcgca gaaaaaaagg atctcaagaa 36300gatcctttga tcttttctac ggggtctgac gctcagtgga acgaaaactc acgttaaggg 36360attttggtca tgagattatc aaaaaggatc ttcacctaga tccttttaaa tcaatctaaa 36420gtatatatga gtaaacttgg tctgacagtt accaatgctt aatcagtgag gcacctatct 36480cagcgatctg tctatttcgt tcatccatag ttgcctgact ccccgtcgtg tagataacta 36540cgatacggga gggcttacca tctggcccca gtgctgcaat gataccgcga gacccacgct 36600caccggctcc agatttatca gcaataaacc agccagccgg aagggccgag cgcagaagtg 36660gtcctgcaac tttatccgcc tccatccagt ctattaattg ttgccgggaa gctagagtaa 36720gtagttcgcc agttaatagt ttgcgcaacg ttgttgccat tgntgcaggc atcgtggtgt 36780cacgctcgtc gtttggtatg gcttcattca gctccggttc ccaacgatca aggcgagtta 36840catgatcccc catgttgtgc aaaaaagcgg ttagctcctt cggtcctccg atcgttgtca 36900gaagtaagtt ggccgcagtg ttatcactca tggttatggc agcactgcat aattctctta 36960ctgtcatgcc atccgtaaga tgcttttctg tgactggtga gtactcaacc aagtcattct 37020gagaatagtg tatgcggcga ccgagttgct cttgcccggc gtcaacacgg gataataccg 37080cgccacatag cagaacttta aaagtgctca tcattggaaa acgttcttcg gggcgaaaac 37140tctcaaggat cttaccgctg ttgagatcca gttcgatgta acccactcgt gcacccaact 37200gatcttcagc atcttttact ttcaccagcg tttctgggtg agcaaaaaca ggaaggcaaa 37260atgccgcaaa aaagggaata agggcgacac ggaaatgttg aatactcata ctcttccttt 37320ttcaatatta ttgaagcatt tatcagggtt attgtctcat gagcggatac atatttgaat 37380gtatttagaa aaataaacaa ataggggttc cgcgcacatt tccccgaaaa gtgccacctg 37440acgtctaaga aaccattatt atcatgacat taacctataa aaataggcgt atcacgaggc 37500cctttcgtct tcaaggatcc gaattcccgg gagagctcga tatcgcatgc ggatttaaat 37560taattaa 37567 89 5038 DNA Artificial Sequence pIB/V5-His-DEST 89catgatgata aacaatgtat ggtgctaatg ttgcttcaac aacaattctg ttgaactgtg 60ttttcatgtt tgccaacaag cacctttata ctcggtggcc tccccaccac caactttttt 120gcactgcaaa aaaacacgct tttgcacgcg ggcccataca tagtacaaac tctacgtttc 180gtagactatt ttacataaat agtctacacc gttgtatacg ctccaaatac actaccacac 240attgaacctt tttgcagtgc aaaaaagtac gtgtcggcag tcacgtaggc cggccttatc 300gggtcgcgtc ctgtcacgta cgaatcacat tatcggaccg gacgagtgtt gtcttatcgt 360gacaggacgc cagcttcctg tgttgctaac cgcagccgga cgcaactcct tatcggaaca 420ggacgcgcct ccatatcagc cgcgcgttat ctcatgcacg tgaccggaca cgaggcgccc 480gtcccgctta tcgcgcctat aaatacagcc cgcaacgatc tggtaaacac agttgaacag 540catctgttcg aatttaaagc ttgatatcga attcctgcag cccagcgctg gatcctcgat 600cacaagtttg tacaaaaaag ctgaacgaga aacgtaaaat gatataaata tcaatatatt 660aaattagatt ttgcataaaa aacagactac ataatactgt aaaacacaac atatccagtc 720actatggcgg ccgcattagg caccccaggc tttacacttt atgcttccgg ctcgtataat 780gtgtggattt tgagttagga tccgtcgaga ttttcaggag ctaaggaagc taaaatggag 840aaaaaaatca ctggatatac caccgttgat atatcccaat ggcatcgtaa agaacatttt 900gaggcatttc agtcagttgc tcaatgtacc tataaccaga ccgttcagct ggatattacg 960gcctttttaa agaccgtaaa gaaaaataag cacaagtttt atccggcctt tattcacatt 1020cttgcccgcc tgatgaatgc tcatccggaa ttccgtatgg caatgaaaga cggtgagctg 1080gtgatatggg atagtgttca cccttgttac accgttttcc atgagcaaac tgaaacgttt 1140tcatcgctct ggagtgaata ccacgacgat ttccggcagt ttctacacat atattcgcaa 1200gatgtggcgt gttacggtga aaacctggcc tatttcccta aagggtttat tgagaatatg 1260tttttcgtct cagccaatcc ctgggtgagt ttcaccagtt ttgatttaaa cgtggccaat 1320atggacaact tcttcgcccc cgttttcacc atgggcaaat attatacgca aggcgacaag 1380gtgctgatgc cgctggcgat tcaggttcat catgccgttt gtgatggctt ccatgtcggc 1440agaatgctta atgaattaca acagtactgc gatgagtggc agggcggggc gtaaacgcgt 1500ggatccggct tactaaaagc cagataacag tatgcgtatt tgcgcgctga tttttgcggt 1560ataagaatat atactgatat gtatacccga agtatgtcaa aaagaggtat gctatgaagc 1620agcgtattac agtgacagtt gacagcgaca gctatcagtt gctcaaggca tatatgatgt 1680caatatctcc ggtctggtaa gcacaaccat gcagaatgaa gcccgtcgtc tgcgtgccga 1740acgctggaaa gcggaaaatc aggaagggat ggctgaggtc gcccggttta ttgaaatgaa 1800cggctctttt gctgacgaga acaggggctg gtgaaatgca gtttaaggtt tacacctata 1860aaagagagag ccgttatcgt ctgtttgtgg atgtacagag tgatattatt gacacgcccg 1920ggcgacggat ggtgatcccc ctggccagtg cacgtctgct gtcagataaa gtctcccgtg 1980aactttaccc ggtggtgcat atcggggatg aaagctggcg catgatgacc accgatatgg 2040ccagtgtgcc ggtctccgtt atcggggaag aagtggctga tctcagccac cgcgaaaatg 2100acatcaaaaa cgccattaac ctgatgttct ggggaatata aatgtcaggc tcccttatac 2160acagccagtc tgcaggtcga ccatagtgac tggatatgtt gtgttttaca gtattatgta 2220gtctgttttt tatgcaaaat ctaatttaat atattgatat ttatatcatt ttacgtttct 2280cgttcagctt tcttgtacaa agtggtgatc gacccgggtc tagagggccc gcggttcgaa 2340ggtaagccta tccctaaccc tctcctcggt ctcgattcta cgcgtaccgg tcatcatcac 2400catcaccatt gagtttatct gactaaatct tagtttgtat tgtcatgttt taatacaata 2460tgttatgttt aaatatgttt ttaataaatt ttataaaata atttcaactt ttattgtaac 2520aacattgtcc atttacacac tcctttcaag cgcgtgggat cgatgctcac tcaaaggcgg 2580taatacggtt atccacagaa tcaggggata acgcaggaaa gaacatgtga gcaaaaggcc 2640agcaaaaggc caggaaccgt aaaaaggccg cgttgctggc gtttttccat aggctccgcc 2700cccctgacga gcatcacaaa aatcgacgct caagtcagag gtggcgaaac ccgacaggac 2760tataaagata ccaggcgttt ccccctggaa gctccctcgt gcgctctcct gttccgaccc 2820tgccgcttac cggatacctg tccgcctttc tcccttcggg aagcgtggcg ctttctcata 2880gctcacgctg taggtatctc agttcggtgt aggtcgttcg ctccaagctg ggctgtgtgc 2940acgaaccccc cgttcagccc gaccgctgcg ccttatccgg taactatcgt cttgagtcca 3000acccggtaag acacgactta tcgccactgg cagcagccac tggtaacagg attagcagag 3060cgaggtatgt aggcggtgct acagagttct tgaagtggtg gcctaactac ggctacacta 3120gaagaacagt atttggtatc tgcgctctgc tgaagccagt taccttcgga aaaagagttg 3180gtagctcttg atccggcaaa caaaccaccg ctggtagcgg tggttttttt gtttgcaagc 3240agcagattac gcgcagaaaa aaaggatctc aagaagatcc tttgatcttt tctacggggt 3300ctgacgctca gtggaacgaa aactcacgtt aagggatttt ggtcatgccc ttgttccgaa 3360gggttgtgtc acgtaggcca gataacggtc gggtatataa gatgcctcaa tgctactagt 3420aaatcagtca caccaaggct tcaataagga acacacaagc aagccctttg agtcaagggc 3480tgccgggctg cagcacgtgt tgacaattaa tcatcggcat agtatatcgg catagtataa 3540tacgacaagg tgaggaacta aaccatggcc aagcctttgt ctcaagaaga atccaccctc 3600attgaaagag caacggctac aatcaacagc atccccatct ctgaagacta cagcgtcgcc 3660ggcgcagctc tctctagcga cggccgcatc ttcactggtg tcaatgtata tcattttact 3720gggggacctt gcgcagaact cgtggtgctg ggcactgctg ctgctgcggc agctggcaac 3780ctgacttgta tcgtcgcgat cggaaatgag aacaggggca tcttgagccc ctgcggacgg 3840tgccgacagg ttcttctcga tctgcatcct gggatcaaag ccatagtgaa ggacagtgat 3900ggacagccga cggcagttgg gattcgtgaa ttgctgccct ctggttatgt gtgggagggc 3960taagcacttc gtggccgagg agcaggactg acacgtcccg ggagatctgc atgtctacta 4020aactcacaaa ttagagcttc aatttaatta tatcagttat tacccattga aaaaggaaga 4080gtatgagtat tcaacatttc cgtgtcgccc ttattccctt ttttgcggca ttttgccttc 4140ctgtttttgc tcacccagaa acgctggtga aagtaaaaga tgctgaagat cagttgggtg 4200cacgagtggg ttacatcgaa ctggatctca acagcggtaa gatccttgag agttttcgcc 4260ccgaagaacg ttttccaatg atgagcactt ttaaagttct gctatgtggc gcggtattat 4320cccgtattga cgccgggcaa gagcaactcg gtcgccgcat acactattct cagaatgact 4380tggttgagta ctcaccagtc acagaaaagc atcttacgga tggcatgaca gtaagagaat 4440tatgcagtgc tgccataacc atgagtgata acactgcggc caacttactt ctgacaacga 4500tcggaggacc gaaggagcta accgcttttt tgcacaacat gggggatcat gtaactcgcc 4560ttgatcgttg ggaaccggag ctgaatgaag ccataccaaa cgacgagcgt gacaccacga 4620tgcctgtagc aatggcaaca acgttgcgca aactattaac tggcgaacta cttactctag 4680cttcccggca acaattaata gactggatgg aggcggataa agttgcagga ccacttctgc 4740gctcggccct tccggctggc tggtttattg ctgataaatc tggagccggt gagcgtgggt 4800ctcgcggtat cattgcagca ctggggccag atggtaagcc ctcccgtatc gtagttatct 4860acacgacggg gagtcaggca actatggatg aacgaaatag acagatcgct gagataggtg 4920cctcactgat taagcattgg taactgtcag accaagttta ctcatatata ctttagattg 4980atttaaaact tcatttttaa tttaaaagga tctaggtgaa gatccttttt gataatct 5038 905693 DNA Artificial Sequence V5-His DEST cassette 90 ataagtattttactgttttc gtaacagttt tgtaataaaa aaacctataa atattccgga 60 ttattcataccgtcccacca tcgggcgcgg atccccgggt accgatatca caagtttgta 120 caaaaaagctgaacgagaaa cgtaaaatga tataaatatc aatatattaa attagatttt 180 gcataaaaaacagactacat aatactgtaa aacacaacat atccagtcac tatggcggcc 240 gctccctaacccacggggcc cgtggctatg gcagggcttg ccgccccgac gttggctgcg 300 agccctgggccttcacccga acttgggggt tggggtgggg aaaaggaaga aacgcgggcg 360 tattggtcccaatggggtct cggtggggta tcgacagagt gccagccctg ggaccgaacc 420 ccgcgtttatgaacaaacga cccaacaccc gtgcgtttta ttctgtcttt ttattgccgt 480 catagcgcgggttccttccg gtattgtctc cttccgtgtt tcagttagcc tcccccatct 540 cccgggcaaacgtgcgcgcc aggtcgcaga tcgtcggtat ggagcctggg gtggtgacgt 600 gggtctggaccatcccggag gtaagttgca gcagggcgtc ccggcagccg gcgggcgatt 660 ggtcgtaatccaggataaag acatgcatgg gacggaggcg tttggccaag acgtccaaag 720 cccaggcaaacacgttatac aggtcgccgt tgggggccag caactcgggg gcccgaaaca 780 gggtaaataacgtgtccccg atatggggtc gtgggcccgc gttgctctgg ggctcggcac 840 cctggggcggcacggccgcc cccgaaagct gtccccaatc ctcccgccac gacccgccgc 900 cctgcagataccgcaccgta ttggcaagca gcccataaac gcggcgaatc gcggccagca 960 tagccaggtcaagccgctcg ccggggcgct ggcgtttggc caggcggtcg atgtgtctgt 1020 cctccggaagggcccccaac acgatgtttg tgccgggcaa ggtcggcggg atgagggcca 1080 cgaacgccagcacggcctgg ggggtcatgc tgcccataag gtatcgcgcg gccgggtagc 1140 acaggagggcggcgatggga tggcggtcga agatgagggt gagggccggg ggcggggcat 1200 gtgagctcccagcctccccc ccgatatgag gagccagaac ggcgtcggtc acggcataag 1260 gcatgcccattgttatctgg gcgcttgtca ttaccaccgc cgcgtccccg gccgatatct 1320 caccctggtcgaggcggtgt tgtgtggtgt agatgttcgc gattgtctcg gaagccccca 1380 acacccgccagtaagtcatc ggctcgggta cgtagacgat atcgtcgcgc gaacccaggg 1440 ccaccagcagttgcgtggtg gtggttttcc ccatcccgtg gggaccgtct atataaaccc 1500 gcagtagcgtgggcattttc tgctccaggc ggacttccgt ggctttttgt tgccggcgag 1560 ggcgcaacgccgtacgtcgg ttgttatggc cgcgagaacg cgcagcctgg tcgaacgcag 1620 acgcgtgttgatggcagggg tacgaagcca tagatcccgt tatcaattac ttatactatc 1680 cggcgcgcaagcgagcgtgt gcgccggagc acaattgata ctgatttacg agttgggcaa 1740 acgggctttatatagcctgt cccctccaca gccctagtgc cgtgcgcaaa gtgcctacgt 1800 gaccaggctctcctacgcat atacaatctt atctctatag ataaggtttc catatataaa 1860 gcctctcgatggctgaacgt gcacagtatc gtgttgattt ctgagtgcta actaacagtt 1920 acaatgaaccgtttttttcg agagaataac atttttgacg cgccaaggac cgggggcaag 1980 ggtcgtgccaaatctttgcc agcgcctgcc gccaactcgc cgccgtcgcc tgttcgtccg 2040 ccgccaaaatctaacatcaa accacctacg cgcatctctc cgcctaaaca gcctatgtgc 2100 acctctccggccaagccgtt ggagcacagc agcattgtaa gtaaaaaacc agtcgtcaac 2160 agaaaagatggatattttgt gccgcccgag tttgggaaca agtttgaagg tttgcccgcg 2220 tacagcgacaaactggattt caaacaagag cgcgatctac gtacctgcag gcccgggctc 2280 aacccaacacaatatattat agttaaataa gaattattat caaatcattt gtatattaat 2340 taaaatactatactgtaaat tacattttat ttacaattca ctctaga atg acc atg 2396 Met Thr Met 1att acg gat tca ctg gcc gtc gtt tta caa cgt cgt gac tgg gaa aac 2444 IleThr Asp Ser Leu Ala Val Val Leu Gln Arg Arg Asp Trp Glu Asn 5 10 15 cctggc gtt acc caa ctt aat cgc ctt gca gca cat ccc cct ttc gcc 2492 Pro GlyVal Thr Gln Leu Asn Arg Leu Ala Ala His Pro Pro Phe Ala 20 25 30 35 agctgg cgt aat agc gaa gag gcc cgc acc gat cgc cct tcc caa cag 2540 Ser TrpArg Asn Ser Glu Glu Ala Arg Thr Asp Arg Pro Ser Gln Gln 40 45 50 ttg cgcagc ctg aat ggc gaa tgg cgc ttt gcc tgg ttt ccg gca cca 2588 Leu Arg SerLeu Asn Gly Glu Trp Arg Phe Ala Trp Phe Pro Ala Pro 55 60 65 gaa gcg gtgccg gaa agc tgg ctg gag tgc gat ctt cct gag gcc gat 2636 Glu Ala Val ProGlu Ser Trp Leu Glu Cys Asp Leu Pro Glu Ala Asp 70 75 80 act gtc gtc gtcccc tca aac tgg cag atg cac ggt tac gat gcg ccc 2684 Thr Val Val Val ProSer Asn Trp Gln Met His Gly Tyr Asp Ala Pro 85 90 95 atc tac acc aac gtaacc tat ccc att acg gtc aat ccg ccg ttt gtt 2732 Ile Tyr Thr Asn Val ThrTyr Pro Ile Thr Val Asn Pro Pro Phe Val 100 105 110 115 ccc acg gag aatccg acg ggt tgt tac tcg ctc aca ttt aat gtt gat 2780 Pro Thr Glu Asn ProThr Gly Cys Tyr Ser Leu Thr Phe Asn Val Asp 120 125 130 gaa agc tgg ctacag gaa ggc cag acg cga att att ttt gat ggc gtt 2828 Glu Ser Trp Leu GlnGlu Gly Gln Thr Arg Ile Ile Phe Asp Gly Val 135 140 145 aac tcg gcg tttcat ctg tgg tgc aac ggg cgc tgg gtc ggt tac ggc 2876 Asn Ser Ala Phe HisLeu Trp Cys Asn Gly Arg Trp Val Gly Tyr Gly 150 155 160 cag gac agt cgtttg ccg tct gaa ttt gac ctg agc gca ttt tta cgc 2924 Gln Asp Ser Arg LeuPro Ser Glu Phe Asp Leu Ser Ala Phe Leu Arg 165 170 175 gcc gga gaa aaccgc ctc gcg gtg atg gtg ctg cgt tgg agt gac ggc 2972 Ala Gly Glu Asn ArgLeu Ala Val Met Val Leu Arg Trp Ser Asp Gly 180 185 190 195 agt tat ctggaa gat cag gat atg tgg cgg atg agc ggc att ttc cgt 3020 Ser Tyr Leu GluAsp Gln Asp Met Trp Arg Met Ser Gly Ile Phe Arg 200 205 210 gac gtc tcgttg ctg cat aaa ccg act aca caa atc agc gat ttc cat 3068 Asp Val Ser LeuLeu His Lys Pro Thr Thr Gln Ile Ser Asp Phe His 215 220 225 gtt gcc actcgc ttt aat gat gat ttc agc cgc gct gta ctg gag gct 3116 Val Ala Thr ArgPhe Asn Asp Asp Phe Ser Arg Ala Val Leu Glu Ala 230 235 240 gaa gtt cagatg tgc ggc gag ttg cgt gac tac cta cgg gta aca gtt 3164 Glu Val Gln MetCys Gly Glu Leu Arg Asp Tyr Leu Arg Val Thr Val 245 250 255 tct tta tggcag ggt gaa acg cag gtc gcc agc ggc acc gcg cct ttc 3212 Ser Leu Trp GlnGly Glu Thr Gln Val Ala Ser Gly Thr Ala Pro Phe 260 265 270 275 ggc ggtgaa att atc gat gag cgt ggt ggt tat gcc gat cgc gtc aca 3260 Gly Gly GluIle Ile Asp Glu Arg Gly Gly Tyr Ala Asp Arg Val Thr 280 285 290 cta cgtctg aac gtc gaa aac ccg aaa ctg tgg agc gcc gaa atc ccg 3308 Leu Arg LeuAsn Val Glu Asn Pro Lys Leu Trp Ser Ala Glu Ile Pro 295 300 305 aat ctctat cgt gcg gtg gtt gaa ctg cac acc gcc gac ggc acg ctg 3356 Asn Leu TyrArg Ala Val Val Glu Leu His Thr Ala Asp Gly Thr Leu 310 315 320 att gaagca gaa gcc tgc gat gtc ggt ttc cgc gag gtg cgg att gaa 3404 Ile Glu AlaGlu Ala Cys Asp Val Gly Phe Arg Glu Val Arg Ile Glu 325 330 335 aat ggtctg ctg ctg ctg aac ggc aag ccg ttg ctg att cga ggc gtt 3452 Asn Gly LeuLeu Leu Leu Asn Gly Lys Pro Leu Leu Ile Arg Gly Val 340 345 350 355 aaccgt cac gag cat cat cct ctg cat ggt cag gtc atg gat gag cag 3500 Asn ArgHis Glu His His Pro Leu His Gly Gln Val Met Asp Glu Gln 360 365 370 acgatg gtg cag gat atc ctg ctg atg aag cag aac aac ttt aac gcc 3548 Thr MetVal Gln Asp Ile Leu Leu Met Lys Gln Asn Asn Phe Asn Ala 375 380 385 gtgcgc tgt tcg cat tat ccg aac cat ccg ctg tgg tac acg ctg tgc 3596 Val ArgCys Ser His Tyr Pro Asn His Pro Leu Trp Tyr Thr Leu Cys 390 395 400 gaccgc tac ggc ctg tat gtg gtg gat gaa gcc aat att gaa acc cac 3644 Asp ArgTyr Gly Leu Tyr Val Val Asp Glu Ala Asn Ile Glu Thr His 405 410 415 ggcatg gtg cca atg aat cgt ctg acc gat gat ccg cgc tgg cta ccg 3692 Gly MetVal Pro Met Asn Arg Leu Thr Asp Asp Pro Arg Trp Leu Pro 420 425 430 435gcg atg agc gaa cgc gta acg cga atg gtg cag cgc gat cgt aat cac 3740 AlaMet Ser Glu Arg Val Thr Arg Met Val Gln Arg Asp Arg Asn His 440 445 450ccg agt gtg atc atc tgg tcg ctg ggg aat gaa tca ggc cac ggc gct 3788 ProSer Val Ile Ile Trp Ser Leu Gly Asn Glu Ser Gly His Gly Ala 455 460 465aat cac gac gcg ctg tat cgc tgg atc aaa tct gtc gat cct tcc cgc 3836 AsnHis Asp Ala Leu Tyr Arg Trp Ile Lys Ser Val Asp Pro Ser Arg 470 475 480ccg gtg cag tat gaa ggc ggc gga gcc gac acc acg gcc acc gat att 3884 ProVal Gln Tyr Glu Gly Gly Gly Ala Asp Thr Thr Ala Thr Asp Ile 485 490 495att tgc ccg atg tac gcg cgc gtg gat gaa gac cag ccc ttc ccg gct 3932 IleCys Pro Met Tyr Ala Arg Val Asp Glu Asp Gln Pro Phe Pro Ala 500 505 510515 gtg ccg aaa tgg tcc atc aaa aaa tgg ctt tcg cta cct gga gag acg 3980Val Pro Lys Trp Ser Ile Lys Lys Trp Leu Ser Leu Pro Gly Glu Thr 520 525530 cgc ccg ctg atc ctt tgc gaa tac gcc cac gcg atg ggt aac agt ctt 4028Arg Pro Leu Ile Leu Cys Glu Tyr Ala His Ala Met Gly Asn Ser Leu 535 540545 ggc ggt ttc gct aaa tac tgg cag gcg ttt cgt cag tat ccc cgt tta 4076Gly Gly Phe Ala Lys Tyr Trp Gln Ala Phe Arg Gln Tyr Pro Arg Leu 550 555560 cag ggc ggc ttc gtc tgg gac tgg gtg gat cag tcg ctg att aaa tat 4124Gln Gly Gly Phe Val Trp Asp Trp Val Asp Gln Ser Leu Ile Lys Tyr 565 570575 gat gaa aac ggc aac ccg tgg tcg gct tac ggc ggt gat ttt ggc gat 4172Asp Glu Asn Gly Asn Pro Trp Ser Ala Tyr Gly Gly Asp Phe Gly Asp 580 585590 595 acg ccg aac gat cgc cag ttc tgt atg aac ggt ctg gtc ttt gcc gac4220 Thr Pro Asn Asp Arg Gln Phe Cys Met Asn Gly Leu Val Phe Ala Asp 600605 610 cgc acg ccg cat cca gcg ctg acg gaa gca aaa cac cag cag cag ttt4268 Arg Thr Pro His Pro Ala Leu Thr Glu Ala Lys His Gln Gln Gln Phe 615620 625 ttc cag ttc cgt tta tcc ggg caa acc atc gaa gtg acc agc gaa tac4316 Phe Gln Phe Arg Leu Ser Gly Gln Thr Ile Glu Val Thr Ser Glu Tyr 630635 640 ctg ttc cgt cat agc gat aac gag ctc ctg cac tgg atg gtg gcg ctg4364 Leu Phe Arg His Ser Asp Asn Glu Leu Leu His Trp Met Val Ala Leu 645650 655 gat ggt aag ccg ctg gca agc ggt gaa gtg cct ctg gat gtc gct cca4412 Asp Gly Lys Pro Leu Ala Ser Gly Glu Val Pro Leu Asp Val Ala Pro 660665 670 675 caa ggt aaa cag ttg att gaa ctg cct gaa cta ccg cag ccg gagagc 4460 Gln Gly Lys Gln Leu Ile Glu Leu Pro Glu Leu Pro Gln Pro Glu Ser680 685 690 gcc ggg caa ctc tgg ctc aca gta cgc gta gtg caa ccg aac gcgacc 4508 Ala Gly Gln Leu Trp Leu Thr Val Arg Val Val Gln Pro Asn Ala Thr695 700 705 gca tgg tca gaa gcc ggg cac atc agc gcc tgg cag cag tgg cgtctg 4556 Ala Trp Ser Glu Ala Gly His Ile Ser Ala Trp Gln Gln Trp Arg Leu710 715 720 gcg gaa aac ctc agt gtg acg ctc ccc gcc gcg tcc cac gcc atcccg 4604 Ala Glu Asn Leu Ser Val Thr Leu Pro Ala Ala Ser His Ala Ile Pro725 730 735 cat ctg acc acc agc gaa atg gat ttt tgc atc gag ctg ggt aataag 4652 His Leu Thr Thr Ser Glu Met Asp Phe Cys Ile Glu Leu Gly Asn Lys740 745 750 755 cgt tgg caa ttt aac cgc cag tca ggc ttt ctt tca cag atgtgg att 4700 Arg Trp Gln Phe Asn Arg Gln Ser Gly Phe Leu Ser Gln Met TrpIle 760 765 770 ggc gat aaa aaa caa ctg ctg acg ccg ctg cgc gat cag ttcacc cgt 4748 Gly Asp Lys Lys Gln Leu Leu Thr Pro Leu Arg Asp Gln Phe ThrArg 775 780 785 gca ccg ctg gat aac gac att ggc gta agt gaa gcg acc cgcatt gac 4796 Ala Pro Leu Asp Asn Asp Ile Gly Val Ser Glu Ala Thr Arg IleAsp 790 795 800 cct aac gcc tgg gtc gaa cgc tgg aag gcg gcg ggc cat taccag gcc 4844 Pro Asn Ala Trp Val Glu Arg Trp Lys Ala Ala Gly His Tyr GlnAla 805 810 815 gaa gca gcg ttg ttg cag tgc acg gca gat aca ctt gct gatgcg gtg 4892 Glu Ala Ala Leu Leu Gln Cys Thr Ala Asp Thr Leu Ala Asp AlaVal 820 825 830 835 ctg att acg acc gct cac gcg tgg cag cat cag ggg aaaacc tta ttt 4940 Leu Ile Thr Thr Ala His Ala Trp Gln His Gln Gly Lys ThrLeu Phe 840 845 850 atc agc cgg aaa acc tac cgg att gat ggt agt ggt caaatg gcg att 4988 Ile Ser Arg Lys Thr Tyr Arg Ile Asp Gly Ser Gly Gln MetAla Ile 855 860 865 acc gtt gat gtt gaa gtg gcg agc gat aca ccg cat ccggcg cgg att 5036 Thr Val Asp Val Glu Val Ala Ser Asp Thr Pro His Pro AlaArg Ile 870 875 880 ggc ctg aac tgc cag ctg gcg cag gta gca gag cgg gtaaac tgg ctc 5084 Gly Leu Asn Cys Gln Leu Ala Gln Val Ala Glu Arg Val AsnTrp Leu 885 890 895 gga tta ggg ccg caa gaa aac tat ccc gac cgc ctt actgcc gcc tgt 5132 Gly Leu Gly Pro Gln Glu Asn Tyr Pro Asp Arg Leu Thr AlaAla Cys 900 905 910 915 ttt gac cgc tgg gat ctg cca ttg tca gac atg tatacc ccg tac gtc 5180 Phe Asp Arg Trp Asp Leu Pro Leu Ser Asp Met Tyr ThrPro Tyr Val 920 925 930 ttc ccg agc gaa aac ggt ctg cgc tgc ggg acg cgcgaa ttg aat tat 5228 Phe Pro Ser Glu Asn Gly Leu Arg Cys Gly Thr Arg GluLeu Asn Tyr 935 940 945 ggc cca cac cag tgg cgc ggc gac ttc cag ttc aacatc agc cgc tac 5276 Gly Pro His Gln Trp Arg Gly Asp Phe Gln Phe Asn IleSer Arg Tyr 950 955 960 agt caa cag caa ctg atg gaa acc agc cat cgc catctg ctg cac gcg 5324 Ser Gln Gln Gln Leu Met Glu Thr Ser His Arg His LeuLeu His Ala 965 970 975 gaa gaa ggc aca tgg ctg aat atc gac ggt ttc catatg ggg att ggt 5372 Glu Glu Gly Thr Trp Leu Asn Ile Asp Gly Phe His MetGly Ile Gly 980 985 990 995 ggc gac gac tcc tgg agc ccg tca gta tcg gcggaa ttc cag ctg 5417 Gly Asp Asp Ser Trp Ser Pro Ser Val Ser Ala Glu PheGln Leu 1000 1005 1010 agc gcc ggt cgc tac cat tac cag ttg gtc tgg tgtcaa aaa taa 5462 Ser Ala Gly Arg Tyr His Tyr Gln Leu Val Trp Cys Gln Lys1015 1020 tgactgcagg tcgaccatag tgactggata tgttgtgttt tacagtattatgtagtctgt 5522 tttttatgca aaatctaatt taatatattg atatttatat cattttacgtttctcgttca 5582 gctttcttgt acaaagtggt gagaatgaat gaagatctg ggg aag cctatc cct 5636 Gly Lys Pro Ile Pro 1025 aac cct ctc ctc ggt ctc gat tctacg cgt acc ggt cat cat cac 5681 Asn Pro Leu Leu Gly Leu Asp Ser Thr ArgThr Gly His His His 1030 1035 1040 cat cac cat tga 5693 His His His 104591 1024 PRT Artificial Sequence V5-His DEST cassette 91 Met Thr Met IleThr Asp Ser Leu Ala Val Val Leu Gln Arg Arg Asp 1 5 10 15 Trp Glu AsnPro Gly Val Thr Gln Leu Asn Arg Leu Ala Ala His Pro 20 25 30 Pro Phe AlaSer Trp Arg Asn Ser Glu Glu Ala Arg Thr Asp Arg Pro 35 40 45 Ser Gln GlnLeu Arg Ser Leu Asn Gly Glu Trp Arg Phe Ala Trp Phe 50 55 60 Pro Ala ProGlu Ala Val Pro Glu Ser Trp Leu Glu Cys Asp Leu Pro 65 70 75 80 Glu AlaAsp Thr Val Val Val Pro Ser Asn Trp Gln Met His Gly Tyr 85 90 95 Asp AlaPro Ile Tyr Thr Asn Val Thr Tyr Pro Ile Thr Val Asn Pro 100 105 110 ProPhe Val Pro Thr Glu Asn Pro Thr Gly Cys Tyr Ser Leu Thr Phe 115 120 125Asn Val Asp Glu Ser Trp Leu Gln Glu Gly Gln Thr Arg Ile Ile Phe 130 135140 Asp Gly Val Asn Ser Ala Phe His Leu Trp Cys Asn Gly Arg Trp Val 145150 155 160 Gly Tyr Gly Gln Asp Ser Arg Leu Pro Ser Glu Phe Asp Leu SerAla 165 170 175 Phe Leu Arg Ala Gly Glu Asn Arg Leu Ala Val Met Val LeuArg Trp 180 185 190 Ser Asp Gly Ser Tyr Leu Glu Asp Gln Asp Met Trp ArgMet Ser Gly 195 200 205 Ile Phe Arg Asp Val Ser Leu Leu His Lys Pro ThrThr Gln Ile Ser 210 215 220 Asp Phe His Val Ala Thr Arg Phe Asn Asp AspPhe Ser Arg Ala Val 225 230 235 240 Leu Glu Ala Glu Val Gln Met Cys GlyGlu Leu Arg Asp Tyr Leu Arg 245 250 255 Val Thr Val Ser Leu Trp Gln GlyGlu Thr Gln Val Ala Ser Gly Thr 260 265 270 Ala Pro Phe Gly Gly Glu IleIle Asp Glu Arg Gly Gly Tyr Ala Asp 275 280 285 Arg Val Thr Leu Arg LeuAsn Val Glu Asn Pro Lys Leu Trp Ser Ala 290 295 300 Glu Ile Pro Asn LeuTyr Arg Ala Val Val Glu Leu His Thr Ala Asp 305 310 315 320 Gly Thr LeuIle Glu Ala Glu Ala Cys Asp Val Gly Phe Arg Glu Val 325 330 335 Arg IleGlu Asn Gly Leu Leu Leu Leu Asn Gly Lys Pro Leu Leu Ile 340 345 350 ArgGly Val Asn Arg His Glu His His Pro Leu His Gly Gln Val Met 355 360 365Asp Glu Gln Thr Met Val Gln Asp Ile Leu Leu Met Lys Gln Asn Asn 370 375380 Phe Asn Ala Val Arg Cys Ser His Tyr Pro Asn His Pro Leu Trp Tyr 385390 395 400 Thr Leu Cys Asp Arg Tyr Gly Leu Tyr Val Val Asp Glu Ala AsnIle 405 410 415 Glu Thr His Gly Met Val Pro Met Asn Arg Leu Thr Asp AspPro Arg 420 425 430 Trp Leu Pro Ala Met Ser Glu Arg Val Thr Arg Met ValGln Arg Asp 435 440 445 Arg Asn His Pro Ser Val Ile Ile Trp Ser Leu GlyAsn Glu Ser Gly 450 455 460 His Gly Ala Asn His Asp Ala Leu Tyr Arg TrpIle Lys Ser Val Asp 465 470 475 480 Pro Ser Arg Pro Val Gln Tyr Glu GlyGly Gly Ala Asp Thr Thr Ala 485 490 495 Thr Asp Ile Ile Cys Pro Met TyrAla Arg Val Asp Glu Asp Gln Pro 500 505 510 Phe Pro Ala Val Pro Lys TrpSer Ile Lys Lys Trp Leu Ser Leu Pro 515 520 525 Gly Glu Thr Arg Pro LeuIle Leu Cys Glu Tyr Ala His Ala Met Gly 530 535 540 Asn Ser Leu Gly GlyPhe Ala Lys Tyr Trp Gln Ala Phe Arg Gln Tyr 545 550 555 560 Pro Arg LeuGln Gly Gly Phe Val Trp Asp Trp Val Asp Gln Ser Leu 565 570 575 Ile LysTyr Asp Glu Asn Gly Asn Pro Trp Ser Ala Tyr Gly Gly Asp 580 585 590 PheGly Asp Thr Pro Asn Asp Arg Gln Phe Cys Met Asn Gly Leu Val 595 600 605Phe Ala Asp Arg Thr Pro His Pro Ala Leu Thr Glu Ala Lys His Gln 610 615620 Gln Gln Phe Phe Gln Phe Arg Leu Ser Gly Gln Thr Ile Glu Val Thr 625630 635 640 Ser Glu Tyr Leu Phe Arg His Ser Asp Asn Glu Leu Leu His TrpMet 645 650 655 Val Ala Leu Asp Gly Lys Pro Leu Ala Ser Gly Glu Val ProLeu Asp 660 665 670 Val Ala Pro Gln Gly Lys Gln Leu Ile Glu Leu Pro GluLeu Pro Gln 675 680 685 Pro Glu Ser Ala Gly Gln Leu Trp Leu Thr Val ArgVal Val Gln Pro 690 695 700 Asn Ala Thr Ala Trp Ser Glu Ala Gly His IleSer Ala Trp Gln Gln 705 710 715 720 Trp Arg Leu Ala Glu Asn Leu Ser ValThr Leu Pro Ala Ala Ser His 725 730 735 Ala Ile Pro His Leu Thr Thr SerGlu Met Asp Phe Cys Ile Glu Leu 740 745 750 Gly Asn Lys Arg Trp Gln PheAsn Arg Gln Ser Gly Phe Leu Ser Gln 755 760 765 Met Trp Ile Gly Asp LysLys Gln Leu Leu Thr Pro Leu Arg Asp Gln 770 775 780 Phe Thr Arg Ala ProLeu Asp Asn Asp Ile Gly Val Ser Glu Ala Thr 785 790 795 800 Arg Ile AspPro Asn Ala Trp Val Glu Arg Trp Lys Ala Ala Gly His 805 810 815 Tyr GlnAla Glu Ala Ala Leu Leu Gln Cys Thr Ala Asp Thr Leu Ala 820 825 830 AspAla Val Leu Ile Thr Thr Ala His Ala Trp Gln His Gln Gly Lys 835 840 845Thr Leu Phe Ile Ser Arg Lys Thr Tyr Arg Ile Asp Gly Ser Gly Gln 850 855860 Met Ala Ile Thr Val Asp Val Glu Val Ala Ser Asp Thr Pro His Pro 865870 875 880 Ala Arg Ile Gly Leu Asn Cys Gln Leu Ala Gln Val Ala Glu ArgVal 885 890 895 Asn Trp Leu Gly Leu Gly Pro Gln Glu Asn Tyr Pro Asp ArgLeu Thr 900 905 910 Ala Ala Cys Phe Asp Arg Trp Asp Leu Pro Leu Ser AspMet Tyr Thr 915 920 925 Pro Tyr Val Phe Pro Ser Glu Asn Gly Leu Arg CysGly Thr Arg Glu 930 935 940 Leu Asn Tyr Gly Pro His Gln Trp Arg Gly AspPhe Gln Phe Asn Ile 945 950 955 960 Ser Arg Tyr Ser Gln Gln Gln Leu MetGlu Thr Ser His Arg His Leu 965 970 975 Leu His Ala Glu Glu Gly Thr TrpLeu Asn Ile Asp Gly Phe His Met 980 985 990 Gly Ile Gly Gly Asp Asp SerTrp Ser Pro Ser Val Ser Ala Glu Phe 995 1000 1005 Gln Leu Ser Ala GlyArg Tyr His Tyr Gln Leu Val Trp Cys Gln 1010 1015 1020 Lys 92 23 PRTArtificial Sequence V5-His DEST cassette 92 Gly Lys Pro Ile Pro Asn ProLeu Leu Gly Leu Asp Ser Thr Arg Thr 1 5 10 15 Gly His His His His HisHis 20 93 376 PRT Herpesvirus sp. 93 Met Ala Ser Tyr Pro Cys His Gln HisAla Ser Ala Phe Asp Gln Ala 1 5 10 15 Ala Arg Ser Arg Gly His Asn AsnArg Arg Thr Ala Leu Arg Pro Arg 20 25 30 Arg Gln Gln Lys Ala Thr Glu ValArg Leu Glu Gln Lys Met Pro Thr 35 40 45 Leu Leu Arg Val Tyr Ile Asp GlyPro His Gly Met Gly Lys Thr Thr 50 55 60 Thr Thr Gln Leu Leu Val Ala LeuGly Ser Arg Asp Asp Ile Val Tyr 65 70 75 80 Val Pro Glu Pro Met Thr TyrTrp Arg Val Leu Gly Ala Ser Glu Thr 85 90 95 Ile Ala Asn Ile Tyr Thr ThrGln His Arg Leu Asp Gln Gly Glu Ile 100 105 110 Ser Ala Gly Asp Ala AlaVal Val Met Thr Ser Ala Gln Ile Thr Met 115 120 125 Gly Met Pro Tyr AlaVal Thr Asp Ala Val Leu Ala Pro His Ile Gly 130 135 140 Gly Glu Ala GlySer Ser His Ala Pro Pro Pro Ala Leu Thr Leu Ile 145 150 155 160 Phe AspArg His Pro Ile Ala Ala Leu Leu Cys Tyr Pro Ala Ala Arg 165 170 175 TyrLeu Met Gly Ser Met Thr Pro Gln Ala Val Leu Ala Phe Val Ala 180 185 190Leu Ile Pro Pro Thr Leu Pro Gly Thr Asn Ile Val Leu Gly Ala Leu 195 200205 Pro Glu Asp Arg His Ile Asp Arg Leu Ala Lys Arg Gln Arg Pro Gly 210215 220 Glu Arg Leu Asp Leu Ala Met Leu Ala Ala Ile Arg Arg Val Tyr Gly225 230 235 240 Leu Leu Ala Asn Thr Val Arg Tyr Leu Gln Gly Gly Gly SerTrp Arg 245 250 255 Glu Asp Trp Gly Gln Leu Ser Gly Ala Ala Val Pro ProGln Gly Ala 260 265 270 Glu Pro Gln Ser Asn Ala Gly Pro Arg Pro His IleGly Asp Thr Leu 275 280 285 Phe Thr Leu Phe Arg Ala Pro Glu Leu Leu AlaPro Asn Gly Asp Leu 290 295 300 Tyr Asn Val Phe Ala Trp Ala Leu Asp ValLeu Ala Lys Arg Leu Arg 305 310 315 320 Pro Met His Val Phe Ile Leu AspTyr Asp Gln Ser Pro Ala Gly Cys 325 330 335 Arg Asp Ala Leu Leu Gln LeuThr Ser Gly Met Val Gln Thr His Val 340 345 350 Thr Thr Pro Gly Ser IlePro Thr Ile Cys Asp Leu Ala Arg Thr Phe 355 360 365 Ala Arg Glu Met GlyGlu Ala Asn 370 375 94 5763 DNA Artificial Sequence Mel/V5-His DESTcassette 94 ataagtattt tactgttttc gtaacagttt tgtaataaaa aaacctataaatattccgga 60 ttattcatac cgtcccacca tcgggcgcgg atcctataaa t atg aaa ttctta gtc 116 Met Lys Phe Leu Val 1 5 aac gtt gcc ctt gtt ttt atg gtc gtatac att tct tac atc tat gcg 164 Asn Val Ala Leu Val Phe Met Val Val TyrIle Ser Tyr Ile Tyr Ala 10 15 20 gcatggtcga atcaaacaag tttgtacaaaaaagctgaac gagaaacgta aaatgatata 224 aatatcaata tattaaatta gattttgcataaaaaacaga ctacataata ctgtaaaaca 284 caacatatcc agtcactatg gcggccgctccctaacccac ggggcccgtg gctatggcag 344 ggcttgccgc cccgacgttg gctgcgagccctgggccttc acccgaactt gggggttggg 404 gtggggaaaa ggaagaaacg cgggcgtattggtcccaatg gggtctcggt ggggtatcga 464 cagagtgcca gccctgggac cgaaccccgcgtttatgaac aaacgaccca acacccgtgc 524 gttttattct gtctttttat tgccgtcatagcgcgggttc cttccggtat tgtctccttc 584 cgtgtttcag ttagcctccc ccatctcccgggcaaacgtg cgcgccaggt cgcagatcgt 644 cggtatggag cctggggtgg tgacgtgggtctggaccatc ccggaggtaa gttgcagcag 704 ggcgtcccgg cagccggcgg gcgattggtcgtaatccagg ataaagacat gcatgggacg 764 gaggcgtttg gccaagacgt ccaaagcccaggcaaacacg ttatacaggt cgccgttggg 824 ggccagcaac tcgggggccc gaaacagggtaaataacgtg tccccgatat ggggtcgtgg 884 gcccgcgttg ctctggggct cggcaccctggggcggcacg gccgcccccg aaagctgtcc 944 ccaatcctcc cgccacgacc cgccgccctgcagataccgc accgtattgg caagcagccc 1004 ataaacgcgg cgaatcgcgg ccagcatagccaggtcaagc cgctcgccgg ggcgctggcg 1064 tttggccagg cggtcgatgt gtctgtcctccggaagggcc cccaacacga tgtttgtgcc 1124 gggcaaggtc ggcgggatga gggccacgaacgccagcacg gcctgggggg tcatgctgcc 1184 cataaggtat cgcgcggccg ggtagcacaggagggcggcg atgggatggc ggtcgaagat 1244 gagggtgagg gccgggggcg gggcatgtgagctcccagcc tcccccccga tatgaggagc 1304 cagaacggcg tcggtcacgg cataaggcatgcccattgtt atctgggcgc ttgtcattac 1364 caccgccgcg tccccggccg atatctcaccctggtcgagg cggtgttgtg tggtgtagat 1424 gttcgcgatt gtctcggaag cccccaacacccgccagtaa gtcatcggct cgggtacgta 1484 gacgatatcg tcgcgcgaac ccagggccaccagcagttgc gtggtggtgg ttttccccat 1544 cccgtgggga ccgtctatat aaacccgcagtagcgtgggc attttctgct ccaggcggac 1604 ttccgtggct ttttgttgcc ggcgagggcgcaacgccgta cgtcggttgt tatggccgcg 1664 agaacgcgca gcctggtcga acgcagacgcgtgttgatgg caggggtacg aagccataga 1724 tcccgttatc aattacttat actatccggcgcgcaagcga gcgtgtgcgc cggagcacaa 1784 ttgatactga tttacgagtt gggcaaacgggctttatata gcctgtcccc tccacagccc 1844 tagtgccgtg cgcaaagtgc ctacgtgaccaggctctcct acgcatatac aatcttatct 1904 ctatagataa ggtttccata tataaagcctctcgatggct gaacgtgcac agtatcgtgt 1964 tgatttctga gtgctaacta acagttacaatgaaccgttt ttttcgagag aataacattt 2024 ttgacgcgcc aaggaccggg ggcaagggtcgtgccaaatc tttgccagcg cctgccgcca 2084 actcgccgcc gtcgcctgtt cgtccgccgccaaaatctaa catcaaacca cctacgcgca 2144 tctctccgcc taaacagcct atgtgcacctctccggccaa gccgttggag cacagcagca 2204 ttgtaagtaa aaaaccagtc gtcaacagaaaagatggata ttttgtgccg cccgagtttg 2264 ggaacaagtt tgaaggtttg cccgcgtacagcgacaaact ggatttcaaa caagagcgcg 2324 atctacgtac ctgcaggccc gggctcaacccaacacaata tattatagtt aaataagaat 2384 tattatcaaa tcatttgtat attaattaaaatactatact gtaaattaca ttttatttac 2444 aattcactct aga atg acc atg att acggat tca ctg gcc gtc gtt tta 2493 Met Thr Met Ile Thr Asp Ser Leu Ala ValVal Leu 25 30 caa cgt cgt gac tgg gaa aac cct ggc gtt acc caa ctt aatcgc ctt 2541 Gln Arg Arg Asp Trp Glu Asn Pro Gly Val Thr Gln Leu Asn ArgLeu 35 40 45 gca gca cat ccc cct ttc gcc agc tgg cgt aat agc gaa gag gcccgc 2589 Ala Ala His Pro Pro Phe Ala Ser Trp Arg Asn Ser Glu Glu Ala Arg50 55 60 65 acc gat cgc cct tcc caa cag ttg cgc agc ctg aat ggc gaa tggcgc 2637 Thr Asp Arg Pro Ser Gln Gln Leu Arg Ser Leu Asn Gly Glu Trp Arg70 75 80 ttt gcc tgg ttt ccg gca cca gaa gcg gtg ccg gaa agc tgg ctg gag2685 Phe Ala Trp Phe Pro Ala Pro Glu Ala Val Pro Glu Ser Trp Leu Glu 8590 95 tgc gat ctt cct gag gcc gat act gtc gtc gtc ccc tca aac tgg cag2733 Cys Asp Leu Pro Glu Ala Asp Thr Val Val Val Pro Ser Asn Trp Gln 100105 110 atg cac ggt tac gat gcg ccc atc tac acc aac gta acc tat ccc att2781 Met His Gly Tyr Asp Ala Pro Ile Tyr Thr Asn Val Thr Tyr Pro Ile 115120 125 acg gtc aat ccg ccg ttt gtt ccc acg gag aat ccg acg ggt tgt tac2829 Thr Val Asn Pro Pro Phe Val Pro Thr Glu Asn Pro Thr Gly Cys Tyr 130135 140 145 tcg ctc aca ttt aat gtt gat gaa agc tgg cta cag gaa ggc cagacg 2877 Ser Leu Thr Phe Asn Val Asp Glu Ser Trp Leu Gln Glu Gly Gln Thr150 155 160 cga att att ttt gat ggc gtt aac tcg gcg ttt cat ctg tgg tgcaac 2925 Arg Ile Ile Phe Asp Gly Val Asn Ser Ala Phe His Leu Trp Cys Asn165 170 175 ggg cgc tgg gtc ggt tac ggc cag gac agt cgt ttg ccg tct gaattt 2973 Gly Arg Trp Val Gly Tyr Gly Gln Asp Ser Arg Leu Pro Ser Glu Phe180 185 190 gac ctg agc gca ttt tta cgc gcc gga gaa aac cgc ctc gcg gtgatg 3021 Asp Leu Ser Ala Phe Leu Arg Ala Gly Glu Asn Arg Leu Ala Val Met195 200 205 gtg ctg cgt tgg agt gac ggc agt tat ctg gaa gat cag gat atgtgg 3069 Val Leu Arg Trp Ser Asp Gly Ser Tyr Leu Glu Asp Gln Asp Met Trp210 215 220 225 cgg atg agc ggc att ttc cgt gac gtc tcg ttg ctg cat aaaccg act 3117 Arg Met Ser Gly Ile Phe Arg Asp Val Ser Leu Leu His Lys ProThr 230 235 240 aca caa atc agc gat ttc cat gtt gcc act cgc ttt aat gatgat ttc 3165 Thr Gln Ile Ser Asp Phe His Val Ala Thr Arg Phe Asn Asp AspPhe 245 250 255 agc cgc gct gta ctg gag gct gaa gtt cag atg tgc ggc gagttg cgt 3213 Ser Arg Ala Val Leu Glu Ala Glu Val Gln Met Cys Gly Glu LeuArg 260 265 270 gac tac cta cgg gta aca gtt tct tta tgg cag ggt gaa acgcag gtc 3261 Asp Tyr Leu Arg Val Thr Val Ser Leu Trp Gln Gly Glu Thr GlnVal 275 280 285 gcc agc ggc acc gcg cct ttc ggc ggt gaa att atc gat gagcgt ggt 3309 Ala Ser Gly Thr Ala Pro Phe Gly Gly Glu Ile Ile Asp Glu ArgGly 290 295 300 305 ggt tat gcc gat cgc gtc aca cta cgt ctg aac gtc gaaaac ccg aaa 3357 Gly Tyr Ala Asp Arg Val Thr Leu Arg Leu Asn Val Glu AsnPro Lys 310 315 320 ctg tgg agc gcc gaa atc ccg aat ctc tat cgt gcg gtggtt gaa ctg 3405 Leu Trp Ser Ala Glu Ile Pro Asn Leu Tyr Arg Ala Val ValGlu Leu 325 330 335 cac acc gcc gac ggc acg ctg att gaa gca gaa gcc tgcgat gtc ggt 3453 His Thr Ala Asp Gly Thr Leu Ile Glu Ala Glu Ala Cys AspVal Gly 340 345 350 ttc cgc gag gtg cgg att gaa aat ggt ctg ctg ctg ctgaac ggc aag 3501 Phe Arg Glu Val Arg Ile Glu Asn Gly Leu Leu Leu Leu AsnGly Lys 355 360 365 ccg ttg ctg att cga ggc gtt aac cgt cac gag cat catcct ctg cat 3549 Pro Leu Leu Ile Arg Gly Val Asn Arg His Glu His His ProLeu His 370 375 380 385 ggt cag gtc atg gat gag cag acg atg gtg cag gatatc ctg ctg atg 3597 Gly Gln Val Met Asp Glu Gln Thr Met Val Gln Asp IleLeu Leu Met 390 395 400 aag cag aac aac ttt aac gcc gtg cgc tgt tcg cattat ccg aac cat 3645 Lys Gln Asn Asn Phe Asn Ala Val Arg Cys Ser His TyrPro Asn His 405 410 415 ccg ctg tgg tac acg ctg tgc gac cgc tac ggc ctgtat gtg gtg gat 3693 Pro Leu Trp Tyr Thr Leu Cys Asp Arg Tyr Gly Leu TyrVal Val Asp 420 425 430 gaa gcc aat att gaa acc cac ggc atg gtg cca atgaat cgt ctg acc 3741 Glu Ala Asn Ile Glu Thr His Gly Met Val Pro Met AsnArg Leu Thr 435 440 445 gat gat ccg cgc tgg cta ccg gcg atg agc gaa cgcgta acg cga atg 3789 Asp Asp Pro Arg Trp Leu Pro Ala Met Ser Glu Arg ValThr Arg Met 450 455 460 465 gtg cag cgc gat cgt aat cac ccg agt gtg atcatc tgg tcg ctg ggg 3837 Val Gln Arg Asp Arg Asn His Pro Ser Val Ile IleTrp Ser Leu Gly 470 475 480 aat gaa tca ggc cac ggc gct aat cac gac gcgctg tat cgc tgg atc 3885 Asn Glu Ser Gly His Gly Ala Asn His Asp Ala LeuTyr Arg Trp Ile 485 490 495 aaa tct gtc gat cct tcc cgc ccg gtg cag tatgaa ggc ggc gga gcc 3933 Lys Ser Val Asp Pro Ser Arg Pro Val Gln Tyr GluGly Gly Gly Ala 500 505 510 gac acc acg gcc acc gat att att tgc ccg atgtac gcg cgc gtg gat 3981 Asp Thr Thr Ala Thr Asp Ile Ile Cys Pro Met TyrAla Arg Val Asp 515 520 525 gaa gac cag ccc ttc ccg gct gtg ccg aaa tggtcc atc aaa aaa tgg 4029 Glu Asp Gln Pro Phe Pro Ala Val Pro Lys Trp SerIle Lys Lys Trp 530 535 540 545 ctt tcg cta cct gga gag acg cgc ccg ctgatc ctt tgc gaa tac gcc 4077 Leu Ser Leu Pro Gly Glu Thr Arg Pro Leu IleLeu Cys Glu Tyr Ala 550 555 560 cac gcg atg ggt aac agt ctt ggc ggt ttcgct aaa tac tgg cag gcg 4125 His Ala Met Gly Asn Ser Leu Gly Gly Phe AlaLys Tyr Trp Gln Ala 565 570 575 ttt cgt cag tat ccc cgt tta cag ggc ggcttc gtc tgg gac tgg gtg 4173 Phe Arg Gln Tyr Pro Arg Leu Gln Gly Gly PheVal Trp Asp Trp Val 580 585 590 gat cag tcg ctg att aaa tat gat gaa aacggc aac ccg tgg tcg gct 4221 Asp Gln Ser Leu Ile Lys Tyr Asp Glu Asn GlyAsn Pro Trp Ser Ala 595 600 605 tac ggc ggt gat ttt ggc gat acg ccg aacgat cgc cag ttc tgt atg 4269 Tyr Gly Gly Asp Phe Gly Asp Thr Pro Asn AspArg Gln Phe Cys Met 610 615 620 625 aac ggt ctg gtc ttt gcc gac cgc acgccg cat cca gcg ctg acg gaa 4317 Asn Gly Leu Val Phe Ala Asp Arg Thr ProHis Pro Ala Leu Thr Glu 630 635 640 gca aaa cac cag cag cag ttt ttc cagttc cgt tta tcc ggg caa acc 4365 Ala Lys His Gln Gln Gln Phe Phe Gln PheArg Leu Ser Gly Gln Thr 645 650 655 atc gaa gtg acc agc gaa tac ctg ttccgt cat agc gat aac gag ctc 4413 Ile Glu Val Thr Ser Glu Tyr Leu Phe ArgHis Ser Asp Asn Glu Leu 660 665 670 ctg cac tgg atg gtg gcg ctg gat ggtaag ccg ctg gca agc ggt gaa 4461 Leu His Trp Met Val Ala Leu Asp Gly LysPro Leu Ala Ser Gly Glu 675 680 685 gtg cct ctg gat gtc gct cca caa ggtaaa cag ttg att gaa ctg cct 4509 Val Pro Leu Asp Val Ala Pro Gln Gly LysGln Leu Ile Glu Leu Pro 690 695 700 705 gaa cta ccg cag ccg gag agc gccggg caa ctc tgg ctc aca gta cgc 4557 Glu Leu Pro Gln Pro Glu Ser Ala GlyGln Leu Trp Leu Thr Val Arg 710 715 720 gta gtg caa ccg aac gcg acc gcatgg tca gaa gcc ggg cac atc agc 4605 Val Val Gln Pro Asn Ala Thr Ala TrpSer Glu Ala Gly His Ile Ser 725 730 735 gcc tgg cag cag tgg cgt ctg gcggaa aac ctc agt gtg acg ctc ccc 4653 Ala Trp Gln Gln Trp Arg Leu Ala GluAsn Leu Ser Val Thr Leu Pro 740 745 750 gcc gcg tcc cac gcc atc ccg catctg acc acc agc gaa atg gat ttt 4701 Ala Ala Ser His Ala Ile Pro His LeuThr Thr Ser Glu Met Asp Phe 755 760 765 tgc atc gag ctg ggt aat aag cgttgg caa ttt aac cgc cag tca ggc 4749 Cys Ile Glu Leu Gly Asn Lys Arg TrpGln Phe Asn Arg Gln Ser Gly 770 775 780 785 ttt ctt tca cag atg tgg attggc gat aaa aaa caa ctg ctg acg ccg 4797 Phe Leu Ser Gln Met Trp Ile GlyAsp Lys Lys Gln Leu Leu Thr Pro 790 795 800 ctg cgc gat cag ttc acc cgtgca ccg ctg gat aac gac att ggc gta 4845 Leu Arg Asp Gln Phe Thr Arg AlaPro Leu Asp Asn Asp Ile Gly Val 805 810 815 agt gaa gcg acc cgc att gaccct aac gcc tgg gtc gaa cgc tgg aag 4893 Ser Glu Ala Thr Arg Ile Asp ProAsn Ala Trp Val Glu Arg Trp Lys 820 825 830 gcg gcg ggc cat tac cag gccgaa gca gcg ttg ttg cag tgc acg gca 4941 Ala Ala Gly His Tyr Gln Ala GluAla Ala Leu Leu Gln Cys Thr Ala 835 840 845 gat aca ctt gct gat gcg gtgctg att acg acc gct cac gcg tgg cag 4989 Asp Thr Leu Ala Asp Ala Val LeuIle Thr Thr Ala His Ala Trp Gln 850 855 860 865 cat cag ggg aaa acc ttattt atc agc cgg aaa acc tac cgg att gat 5037 His Gln Gly Lys Thr Leu PheIle Ser Arg Lys Thr Tyr Arg Ile Asp 870 875 880 ggt agt ggt caa atg gcgatt acc gtt gat gtt gaa gtg gcg agc gat 5085 Gly Ser Gly Gln Met Ala IleThr Val Asp Val Glu Val Ala Ser Asp 885 890 895 aca ccg cat ccg gcg cggatt ggc ctg aac tgc cag ctg gcg cag gta 5133 Thr Pro His Pro Ala Arg IleGly Leu Asn Cys Gln Leu Ala Gln Val 900 905 910 gca gag cgg gta aac tggctc gga tta ggg ccg caa gaa aac tat ccc 5181 Ala Glu Arg Val Asn Trp LeuGly Leu Gly Pro Gln Glu Asn Tyr Pro 915 920 925 gac cgc ctt act gcc gcctgt ttt gac cgc tgg gat ctg cca ttg tca 5229 Asp Arg Leu Thr Ala Ala CysPhe Asp Arg Trp Asp Leu Pro Leu Ser 930 935 940 945 gac atg tat acc ccgtac gtc ttc ccg agc gaa aac ggt ctg cgc tgc 5277 Asp Met Tyr Thr Pro TyrVal Phe Pro Ser Glu Asn Gly Leu Arg Cys 950 955 960 ggg acg cgc gaa ttgaat tat ggc cca cac cag tgg cgc ggc gac ttc 5325 Gly Thr Arg Glu Leu AsnTyr Gly Pro His Gln Trp Arg Gly Asp Phe 965 970 975 cag ttc aac atc agccgc tac agt caa cag caa ctg atg gaa acc agc 5373 Gln Phe Asn Ile Ser ArgTyr Ser Gln Gln Gln Leu Met Glu Thr Ser 980 985 990 cat cgc cat ctg ctgcac gcg gaa gaa ggc aca tgg ctg aat atc gac 5421 His Arg His Leu Leu HisAla Glu Glu Gly Thr Trp Leu Asn Ile Asp 995 1000 1005 ggt ttc cat atgggg att ggt ggc gac gac tcc tgg agc ccg tca 5466 Gly Phe His Met Gly IleGly Gly Asp Asp Ser Trp Ser Pro Ser 1010 1015 1020 gta tcg gcg gaa ttccag ctg agc gcc ggt cgc tac cat tac cag 5511 Val Ser Ala Glu Phe Gln LeuSer Ala Gly Arg Tyr His Tyr Gln 1025 1030 1035 ttg gtc tgg tgt caa aaataa tgactgcagg tcgaccatag tgactggata 5562 Leu Val Trp Cys Gln Lys 10401045 tgttgtgttt tacagtatta tgtagtctgt tttttatgca aaatctaatt taatatattg5622 atatttatat cattttacgt ttctcgttca gctttcttgt acaaagtggt gagaatgaat5682 gaagatctg ggg aag cct atc cct aac cct ctc ctc ggt ctc gat tct 5730Gly Lys Pro Ile Pro Asn Pro Leu Leu Gly Leu Asp Ser 1050 1055 acg cgtacc ggt cat cat cac cat cac cat tga 5763 Thr Arg Thr Gly His His His HisHis His 1060 1065 95 21 PRT Artificial Sequence Mel/V5-His DEST cassette95 Met Lys Phe Leu Val Asn Val Ala Leu Val Phe Met Val Val Tyr Ile 1 510 15 Ser Tyr Ile Tyr Ala 20 96 1024 PRT Artificial Sequence Mel/V5-HisDEST cassette 96 Met Thr Met Ile Thr Asp Ser Leu Ala Val Val Leu Gln ArgArg Asp 1 5 10 15 Trp Glu Asn Pro Gly Val Thr Gln Leu Asn Arg Leu AlaAla His Pro 20 25 30 Pro Phe Ala Ser Trp Arg Asn Ser Glu Glu Ala Arg ThrAsp Arg Pro 35 40 45 Ser Gln Gln Leu Arg Ser Leu Asn Gly Glu Trp Arg PheAla Trp Phe 50 55 60 Pro Ala Pro Glu Ala Val Pro Glu Ser Trp Leu Glu CysAsp Leu Pro 65 70 75 80 Glu Ala Asp Thr Val Val Val Pro Ser Asn Trp GlnMet His Gly Tyr 85 90 95 Asp Ala Pro Ile Tyr Thr Asn Val Thr Tyr Pro IleThr Val Asn Pro 100 105 110 Pro Phe Val Pro Thr Glu Asn Pro Thr Gly CysTyr Ser Leu Thr Phe 115 120 125 Asn Val Asp Glu Ser Trp Leu Gln Glu GlyGln Thr Arg Ile Ile Phe 130 135 140 Asp Gly Val Asn Ser Ala Phe His LeuTrp Cys Asn Gly Arg Trp Val 145 150 155 160 Gly Tyr Gly Gln Asp Ser ArgLeu Pro Ser Glu Phe Asp Leu Ser Ala 165 170 175 Phe Leu Arg Ala Gly GluAsn Arg Leu Ala Val Met Val Leu Arg Trp 180 185 190 Ser Asp Gly Ser TyrLeu Glu Asp Gln Asp Met Trp Arg Met Ser Gly 195 200 205 Ile Phe Arg AspVal Ser Leu Leu His Lys Pro Thr Thr Gln Ile Ser 210 215 220 Asp Phe HisVal Ala Thr Arg Phe Asn Asp Asp Phe Ser Arg Ala Val 225 230 235 240 LeuGlu Ala Glu Val Gln Met Cys Gly Glu Leu Arg Asp Tyr Leu Arg 245 250 255Val Thr Val Ser Leu Trp Gln Gly Glu Thr Gln Val Ala Ser Gly Thr 260 265270 Ala Pro Phe Gly Gly Glu Ile Ile Asp Glu Arg Gly Gly Tyr Ala Asp 275280 285 Arg Val Thr Leu Arg Leu Asn Val Glu Asn Pro Lys Leu Trp Ser Ala290 295 300 Glu Ile Pro Asn Leu Tyr Arg Ala Val Val Glu Leu His Thr AlaAsp 305 310 315 320 Gly Thr Leu Ile Glu Ala Glu Ala Cys Asp Val Gly PheArg Glu Val 325 330 335 Arg Ile Glu Asn Gly Leu Leu Leu Leu Asn Gly LysPro Leu Leu Ile 340 345 350 Arg Gly Val Asn Arg His Glu His His Pro LeuHis Gly Gln Val Met 355 360 365 Asp Glu Gln Thr Met Val Gln Asp Ile LeuLeu Met Lys Gln Asn Asn 370 375 380 Phe Asn Ala Val Arg Cys Ser His TyrPro Asn His Pro Leu Trp Tyr 385 390 395 400 Thr Leu Cys Asp Arg Tyr GlyLeu Tyr Val Val Asp Glu Ala Asn Ile 405 410 415 Glu Thr His Gly Met ValPro Met Asn Arg Leu Thr Asp Asp Pro Arg 420 425 430 Trp Leu Pro Ala MetSer Glu Arg Val Thr Arg Met Val Gln Arg Asp 435 440 445 Arg Asn His ProSer Val Ile Ile Trp Ser Leu Gly Asn Glu Ser Gly 450 455 460 His Gly AlaAsn His Asp Ala Leu Tyr Arg Trp Ile Lys Ser Val Asp 465 470 475 480 ProSer Arg Pro Val Gln Tyr Glu Gly Gly Gly Ala Asp Thr Thr Ala 485 490 495Thr Asp Ile Ile Cys Pro Met Tyr Ala Arg Val Asp Glu Asp Gln Pro 500 505510 Phe Pro Ala Val Pro Lys Trp Ser Ile Lys Lys Trp Leu Ser Leu Pro 515520 525 Gly Glu Thr Arg Pro Leu Ile Leu Cys Glu Tyr Ala His Ala Met Gly530 535 540 Asn Ser Leu Gly Gly Phe Ala Lys Tyr Trp Gln Ala Phe Arg GlnTyr 545 550 555 560 Pro Arg Leu Gln Gly Gly Phe Val Trp Asp Trp Val AspGln Ser Leu 565 570 575 Ile Lys Tyr Asp Glu Asn Gly Asn Pro Trp Ser AlaTyr Gly Gly Asp 580 585 590 Phe Gly Asp Thr Pro Asn Asp Arg Gln Phe CysMet Asn Gly Leu Val 595 600 605 Phe Ala Asp Arg Thr Pro His Pro Ala LeuThr Glu Ala Lys His Gln 610 615 620 Gln Gln Phe Phe Gln Phe Arg Leu SerGly Gln Thr Ile Glu Val Thr 625 630 635 640 Ser Glu Tyr Leu Phe Arg HisSer Asp Asn Glu Leu Leu His Trp Met 645 650 655 Val Ala Leu Asp Gly LysPro Leu Ala Ser Gly Glu Val Pro Leu Asp 660 665 670 Val Ala Pro Gln GlyLys Gln Leu Ile Glu Leu Pro Glu Leu Pro Gln 675 680 685 Pro Glu Ser AlaGly Gln Leu Trp Leu Thr Val Arg Val Val Gln Pro 690 695 700 Asn Ala ThrAla Trp Ser Glu Ala Gly His Ile Ser Ala Trp Gln Gln 705 710 715 720 TrpArg Leu Ala Glu Asn Leu Ser Val Thr Leu Pro Ala Ala Ser His 725 730 735Ala Ile Pro His Leu Thr Thr Ser Glu Met Asp Phe Cys Ile Glu Leu 740 745750 Gly Asn Lys Arg Trp Gln Phe Asn Arg Gln Ser Gly Phe Leu Ser Gln 755760 765 Met Trp Ile Gly Asp Lys Lys Gln Leu Leu Thr Pro Leu Arg Asp Gln770 775 780 Phe Thr Arg Ala Pro Leu Asp Asn Asp Ile Gly Val Ser Glu AlaThr 785 790 795 800 Arg Ile Asp Pro Asn Ala Trp Val Glu Arg Trp Lys AlaAla Gly His 805 810 815 Tyr Gln Ala Glu Ala Ala Leu Leu Gln Cys Thr AlaAsp Thr Leu Ala 820 825 830 Asp Ala Val Leu Ile Thr Thr Ala His Ala TrpGln His Gln Gly Lys 835 840 845 Thr Leu Phe Ile Ser Arg Lys Thr Tyr ArgIle Asp Gly Ser Gly Gln 850 855 860 Met Ala Ile Thr Val Asp Val Glu ValAla Ser Asp Thr Pro His Pro 865 870 875 880 Ala Arg Ile Gly Leu Asn CysGln Leu Ala Gln Val Ala Glu Arg Val 885 890 895 Asn Trp Leu Gly Leu GlyPro Gln Glu Asn Tyr Pro Asp Arg Leu Thr 900 905 910 Ala Ala Cys Phe AspArg Trp Asp Leu Pro Leu Ser Asp Met Tyr Thr 915 920 925 Pro Tyr Val PhePro Ser Glu Asn Gly Leu Arg Cys Gly Thr Arg Glu 930 935 940 Leu Asn TyrGly Pro His Gln Trp Arg Gly Asp Phe Gln Phe Asn Ile 945 950 955 960 SerArg Tyr Ser Gln Gln Gln Leu Met Glu Thr Ser His Arg His Leu 965 970 975Leu His Ala Glu Glu Gly Thr Trp Leu Asn Ile Asp Gly Phe His Met 980 985990 Gly Ile Gly Gly Asp Asp Ser Trp Ser Pro Ser Val Ser Ala Glu Phe 9951000 1005 Gln Leu Ser Ala Gly Arg Tyr His Tyr Gln Leu Val Trp Cys Gln1010 1015 1020 Lys 97 23 PRT Artificial Sequence Mel/V5-His DESTcassette 97 Gly Lys Pro Ile Pro Asn Pro Leu Leu Gly Leu Asp Ser Thr ArgThr 1 5 10 15 Gly His His His His His His 20 98 1021 DNA Unknown AcMNPVORF 25 promoter sequence 98 ggtgtcttca ttagtatgcc aatcacgtac gcaacagtcgcaaaagaaac acacagtttc 60 gtctccgcga cccgtgtaaa aaagtcccgc ttccgcaatgtttgtaatca tgtcacgcaa 120 tgcggcaggc caaaagttaa caaacgtatc catacgcgactgtaaattgg acatgcatct 180 gtacacacac ttgggtttgc cttctttcac tagtacagcgttgatggtaa tgttgtcgcc 240 aaacgattca cgctcggcga tcttgttagc atacgcgcaatacggcgaca aggttacgtg 300 tgcatattca atacactcgt cttcggacca attttttatttctgcttcgc aatactcgca 360 cacaacgtga tcgtcaactt gattgtattt aaacccgttaacgatcaagc tgttaataaa 420 cgccgtgttt tcaatgggat aattttcaaa cgaactatgtctttctatta acatgtcgaa 480 tacgtgttcg gcggtgttgt cgcgaaagtt gtcacacacgctgataaaat aaaacggggg 540 cgtgtcctcg ttcattttag ctcgttaaag ttacggtcaaaatgagcacg tttgcgtcgt 600 tttggtttag cgacacgttt atatggccca gtttggtttttgtttcggcg ttaatgacgt 660 gcactgtgga caaatcgtgt tctaaaacta caaactcgtactcgaaaatg tttgatatgt 720 agttggttag ccgatctatc ttaaaattaa acttttgcaactcgctgata gagcacacgt 780 ccacatactt gtcgataaac ccgttgctca accgcttcaaaacggtgtaa ttttgtagct 840 tgaaaggggc gcatttggaa tgactaaaag gaatatttttcaataaatcg tcagtagtgt 900 acgcaaacgc gttgtctacg cacatgctgg caacagagtcgtccatattt attatatatc 960 ttatattctg tgaaacactt caattagact tgaaccacagcagacagcgc acgtcggtag 1020 c 1021 99 1033 DNA Unknown AcMNPV lef 3promoter 99 ccgagaagaa ggcggtttgt ataaaaccca tttttcgaaa tggttaacaaacttgtttag 60 catttggatc gtttcgtgtt caaacgcgtc gaaaactttt aaaacgcaattgccgccggg 120 acgcaggcaa attaaaatta gctgcgtctc gcacatgatc aaatcaaagttgagacgttc 180 ttgttcgttt tcgcgtccat taacgtcaac cgagccatct gccaacaccagatcgcacgc 240 gttgccacac ttgatgctaa tctcaaatac aacattttta tcaaacacgtcgcctgactt 300 gtcgggcccc gtaatggttg tgaaattttt gcgtttgcgc actgtcggtttgtacacgca 360 caccgagttg tttgtcaacg tgacgccata cgctttgcaa agcgggttcaacgacatggt 420 atagttggca aactcgcccg gtccgccgca caaatccaaa aacgtgtcaacgtgtcggca 480 aacgtgaaac tttttgtcga tctctgatag ttttcgccaa catctaggtctgcgcgttgg 540 gcgtttgtca aataattttg agcgagcgca aaccaccgac ttgctgctgaacgtgttcaa 600 accatctttg agtttattta atttttgctg caacattttt actcttcgtgtcggtcgcaa 660 tgtttgtgtc gaaaaagacg gccaacacgc tcagcaaaac tatacaaataaagaacaaaa 720 atacgtacgc aatattaaca ttgaccgttt gatcgttaaa tcggacgggtctgttcagag 780 ccgctcttat tctctcgttg tacattgtta aagtttttgt ttttaaattgtacacaatcg 840 gcgtgttgta gtcgaaattt tcaaaatcgg ctttttgaaa cattgttctgaacgtgttgt 900 cgagcggcgt gttgctggcc acgtttataa tcaactccct ccacgctaacgaacggtgct 960 ctggcgacac ttcgatttcg tcgccattca gtatttgcca tcggatagattcccacatat 1020 cgacaacagc aat 1033 100 1053 DNA Unknown AcMNPV TLPpromoter 100 tgctagccca attggccact gttgtacgaa atatcgtcgt caacgtgtttgaatacatgt 60 tggcccgtac cgttgggtaa atctatgcat ctggagtcgc cggaacactcgtactggttg 120 tcagagtttc tgatccggtt gatgcacgtt atcagttgtg actcgttattattcaaacat 180 ttgaaatatt gcgtgtcgcc gatatcggcc gttatgtacg tgtgtccggcgccgttaaac 240 gcgcacggat gcgcttccac gcacgacatt aagttgcgat caaatattttattcgcgggg 300 cattcgccca ccacgtggcg cccatttacg cactgcataa actggttgacgagcaaattg 360 gagggaaagt atgatagtat atagccgtct ggcctgtttt cacacaattcgttaacttta 420 cactggccgg tttccgcgtc aaacgtgtaa ttatctggac attcttcgactgcgtgcgct 480 ccgtttgcaa aacacctaag atagaacgtg ggatgataca agtgcgcgttggtagaataa 540 tctttgtcca agtgttggtt caacaccaac gtgtccagca aacgctcgtccatgggataa 600 agaccggcag acttgttgtc gcacggcggc acgggaacac attttagttgtgcgtaatca 660 aagttaaaat atgcggggca tttcatggtc acgtcggcct tgtcgccgctcaaaataaac 720 tcgttgggat tttcatcatt tgctctaacg cgatcgtgta cgattcgatcaacaggttga 780 aatttttgat ttaagaaatc aaaaatttca atccggtcat catgcacgctttcgtgatag 840 gtggaaaggt cgacggtgtt gaaccacgtt acaatataag tgttttgcataatatccgac 900 acgtagccta ttacgtcggg tgtgggttcg tctgcgttgg tgcgcttcacatattcagtc 960 atcacttgga gccgcttggt gaaagtcgtt tcgtcaaatt caaaataaattgccaaatac 1020 attaaagtaa acgctattat aagaaaaaag ctt 1053 101 507 DNAUnknown AcMNPV hr5 sequence 101 gttttacgcg tagaattcta cccgtaaagcgagtttagtt atgagccatg tgcaaaacat 60 gacatcagct tttattttta taacaaatgacatcatttct tgattgtgtt ttacacgtag 120 aattctactc gtaaagcgag ttcagttttgaaaaacaaat gacatcatct ttttgattgt 180 gctttacaag tagaattcta cccgtaaatcaagttcggtt ttgaaaaaca aatgagtcat 240 attgtatgat atcatattgc aaacaaatgactcatcaatc gatcgtgcgt acacgtagaa 300 ttctactcgt aaagcgagtt tatgagccgtgtgcaaaaca tgacatcatc tcgatttgaa 360 aaacaaatga catcatccac tgatcgtgcgttacaagtag aattctactc gtaaagccag 420 ttcggttatg agccgtgtgc aaaacatgacatcagcttat gactcgtact tgattgtgtt 480 ttacgcgtag aattctactc gtaaagc 507102 507 DNA Unknown AcMNPV IE-1 promoter 102 gttttacgcg tagaattctacccgtaaagc gagtttagtt atgagccatg tgcaaaacat 60 gacatcagct tttatttttataacaaatga catcatttct tgattgtgtt ttacacgtag 120 aattctactc gtaaagcgagttcagttttg aaaaacaaat gacatcatct ttttgattgt 180 gctttacaag tagaattctacccgtaaatc aagttcggtt ttgaaaaaca aatgagtcat 240 attgtatgat atcatattgcaaacaaatga ctcatcaatc gatcgtgcgt acacgtagaa 300 ttctactcgt aaagcgagtttatgagccgt gtgcaaaaca tgacatcatc tcgatttgaa 360 aaacaaatga catcatccactgatcgtgcg ttacaagtag aattctactc gtaaagccag 420 ttcggttatg agccgtgtgcaaaacatgac atcagcttat gactcgtact tgattgtgtt 480 ttacgcgtag aattctactcgtaaagc 507 103 1746 DNA Unknown AcMPNV IE-1 coding sequence 103atgacgcaaa ttaattttaa cgcgtcgtac accagcgctt cgacgccgtc ccgagcgtcg 60ttcgacaaca gctattcaga gttttgtgat aaacaaccca acgactattt aagttattat 120aaccatccca ccccggatgg agccgacacg gtgatatctg acagcgagac tgcggcagct 180tcaaactttt tggcaagcgt caactcgtta actgataatg atttagtgga atgtttgctc 240aagaccactg ataatctcga agaagcagtt agttctgctt attattcgga atcccttgag 300cagcctgttg tggagcaacc atcgcccagt tctgcttatc atgcggaatc ttttgagcat 360tctgctggtg tgaaccaacc atcggcaact ggaactaaac ggaagctgga cgaatacttg 420gacaattcac aaggtgtggt gggccagttt aacaaaatta aattgaggcc taaatacaag 480aaaagcacaa ttcaaagctg tgcaaccctt gaacagacaa ttaatcacaa cacgaacatt 540tgcacggtcg cttcaactca agaaattacg cattatttta ctaatgattt tgcgccgtat 600ttaatgcgtt tcgacgacaa cgactacaat tccaacaggt tctccgacca tatgtccgaa 660actggttatt acatgtttgt ggttaaaaaa agtgaagtga agccgtttga aattatattt 720gccaagtacg tgagcaatgt ggtttacgaa tatacaaaca attattacat ggtagataat 780cgcgtgtttg tggtaacttt tgataaaatt aggtttatga tttcgtacaa tttggttaaa 840gaaaccggca tagaaattcc tcattctcaa gatgtgtgca acgacgagac ggctgcacaa 900aattgtaaaa aatgccattt cgtcgatgtg caccacacgt ttaaagctgc tctgacttca 960tattttaatt tagatatgta ttacgcgcaa accacatttg tgactttgtt acaatcgttg 1020ggcgaaagaa aatgtgggtt tcttttgagc aagttgtacg aaatgtatca agataaaaat 1080ttatttactt tgcctattat gcttagtcgt aaagagagta atgaaattga gactgcatct 1140aataatttct ttgtatcgcc gtatgtgagt caaatattaa agtattcgga aagtgtgcag 1200tttcccgaca atcccccaaa caaatatgtg gtggacaatt taaatttaat tgttaacaaa 1260aaaagtacgc tcacgtacaa atacagcagc gtcgctaatc ttttgtttaa taattataaa 1320tatcatgaca atattgcgag taataataac gcagaaaatt taaaaaaggt taagaaggag 1380gacggcagca tgcacattgt cgaacagtat ttgactcaga atgtagataa tgtaaagggt 1440cacaatttta tagtattgtc tttcaaaaac gaggagcgat tgactatagc taagaaaaac 1500aaagagtttt attggatttc tggcgaaatt aaagatgtag acgttagtca agtaattcaa 1560aaatataata gatttaagca tcacatgttt gtaatcggta aagtgaaccg aagagagagc 1620actacattgc acaataattt gttaaaattg ttagctttaa tattacaggg tctggttccg 1680ttgtccgacg ctataacgtt tgcggaacaa aaactaaatt gtaaatataa aaaattcgaa 1740tttaat 1746 104 582 PRT Unknown AcMNPV IE-1 protein sequence 104 Met ThrGln Ile Asn Phe Asn Ala Ser Tyr Thr Ser Ala Ser Thr Pro 1 5 10 15 SerArg Ala Ser Phe Asp Asn Ser Tyr Ser Glu Phe Cys Asp Lys Gln 20 25 30 ProAsn Asp Tyr Leu Ser Tyr Tyr Asn His Pro Thr Pro Asp Gly Ala 35 40 45 AspThr Val Ile Ser Asp Ser Glu Thr Ala Ala Ala Ser Asn Phe Leu 50 55 60 AlaSer Val Asn Ser Leu Thr Asp Asn Asp Leu Val Glu Cys Leu Leu 65 70 75 80Lys Thr Thr Asp Asn Leu Glu Glu Ala Val Ser Ser Ala Tyr Tyr Ser 85 90 95Glu Ser Leu Glu Gln Pro Val Val Glu Gln Pro Ser Pro Ser Ser Ala 100 105110 Tyr His Ala Glu Ser Phe Glu His Ser Ala Gly Val Asn Gln Pro Ser 115120 125 Ala Thr Gly Thr Lys Arg Lys Leu Asp Glu Tyr Leu Asp Asn Ser Gln130 135 140 Gly Val Val Gly Gln Phe Asn Lys Ile Lys Leu Arg Pro Lys TyrLys 145 150 155 160 Lys Ser Thr Ile Gln Ser Cys Ala Thr Leu Glu Gln ThrIle Asn His 165 170 175 Asn Thr Asn Ile Cys Thr Val Ala Ser Thr Gln GluIle Thr His Tyr 180 185 190 Phe Thr Asn Asp Phe Ala Pro Tyr Leu Met ArgPhe Asp Asp Asn Asp 195 200 205 Tyr Asn Ser Asn Arg Phe Ser Asp His MetSer Glu Thr Gly Tyr Tyr 210 215 220 Met Phe Val Val Lys Lys Ser Glu ValLys Pro Phe Glu Ile Ile Phe 225 230 235 240 Ala Lys Tyr Val Ser Asn ValVal Tyr Glu Tyr Thr Asn Asn Tyr Tyr 245 250 255 Met Val Asp Asn Arg ValPhe Val Val Thr Phe Asp Lys Ile Arg Phe 260 265 270 Met Ile Ser Tyr AsnLeu Val Lys Glu Thr Gly Ile Glu Ile Pro His 275 280 285 Ser Gln Asp ValCys Asn Asp Glu Thr Ala Ala Gln Asn Cys Lys Lys 290 295 300 Cys His PheVal Asp Val His His Thr Phe Lys Ala Ala Leu Thr Ser 305 310 315 320 TyrPhe Asn Leu Asp Met Tyr Tyr Ala Gln Thr Thr Phe Val Thr Leu 325 330 335Leu Gln Ser Leu Gly Glu Arg Lys Cys Gly Phe Leu Leu Ser Lys Leu 340 345350 Tyr Glu Met Tyr Gln Asp Lys Asn Leu Phe Thr Leu Pro Ile Met Leu 355360 365 Ser Arg Lys Glu Ser Asn Glu Ile Glu Thr Ala Ser Asn Asn Phe Phe370 375 380 Val Ser Pro Tyr Val Ser Gln Ile Leu Lys Tyr Ser Glu Ser ValGln 385 390 395 400 Phe Pro Asp Asn Pro Pro Asn Lys Tyr Val Val Asp AsnLeu Asn Leu 405 410 415 Ile Val Asn Lys Lys Ser Thr Leu Thr Tyr Lys TyrSer Ser Val Ala 420 425 430 Asn Leu Leu Phe Asn Asn Tyr Lys Tyr His AspAsn Ile Ala Ser Asn 435 440 445 Asn Asn Ala Glu Asn Leu Lys Lys Val LysLys Glu Asp Gly Ser Met 450 455 460 His Ile Val Glu Gln Tyr Leu Thr GlnAsn Val Asp Asn Val Lys Gly 465 470 475 480 His Asn Phe Ile Val Leu SerPhe Lys Asn Glu Glu Arg Leu Thr Ile 485 490 495 Ala Lys Lys Asn Lys GluPhe Tyr Trp Ile Ser Gly Glu Ile Lys Asp 500 505 510 Val Asp Val Ser GlnVal Ile Gln Lys Tyr Asn Arg Phe Lys His His 515 520 525 Met Phe Val IleGly Lys Val Asn Arg Arg Glu Ser Thr Thr Leu His 530 535 540 Asn Asn LeuLeu Lys Leu Leu Ala Leu Ile Leu Gln Gly Leu Val Pro 545 550 555 560 LeuSer Asp Ala Ile Thr Phe Ala Glu Gln Lys Leu Asn Cys Lys Tyr 565 570 575Lys Lys Phe Glu Phe Asn 580 105 8688 DNA Artificial SequencepLenti6/V5-DEST 105 aatgtagtct tatgcaatac tcttgtagtc ttgcaacatggtaacgatga gttagcaaca 60 tgccttacaa ggagagaaaa agcaccgtgc atgccgattggtggaagtaa ggtggtacga 120 tcgtgcctta ttaggaaggc aacagacggg tctgacatggattggacgaa ccactgaatt 180 gccgcattgc agagatattg tatttaagtg cctagctcgatacataaacg ggtctctctg 240 gttagaccag atctgagcct gggagctctc tggctaactagggaacccac tgcttaagcc 300 tcaataaagc ttgccttgag tgcttcaagt agtgtgtgcccgtctgttgt gtgactctgg 360 taactagaga tccctcagac ccttttagtc agtgtggaaaatctctagca gtggcgcccg 420 aacagggact tgaaagcgaa agggaaacca gaggagctctctcgacgcag gactcggctt 480 gctgaagcgc gcacggcaag aggcgagggg cggcgactggtgagtacgcc aaaaattttg 540 actagcggag gctagaagga gagagatggg tgcgagagcgtcagtattaa gcgggggaga 600 attagatcgc gatgggaaaa aattcggtta aggccagggggaaagaaaaa atataaatta 660 aaacatatag tatgggcaag cagggagcta gaacgattcgcagttaatcc tggcctgtta 720 gaaacatcag aaggctgtag acaaatactg ggacagctacaaccatccct tcagacagga 780 tcagaagaac ttagatcatt atataataca gtagcaaccctctattgtgt gcatcaaagg 840 atagagataa aagacaccaa ggaagcttta gacaagatagaggaagagca aaacaaaagt 900 aagaccaccg cacagcaagc ggccgctgat cttcagacctggaggaggag atatgaggga 960 caattggaga agtgaattat ataaatataa agtagtaaaaattgaaccat taggagtagc 1020 acccaccaag gcaaagagaa gagtggtgca gagagaaaaaagagcagtgg gaataggagc 1080 tttgttcctt gggttcttgg gagcagcagg aagcactatgggcgcagcgt caatgacgct 1140 gacggtacag gccagacaat tattgtctgg tatagtgcagcagcagaaca atttgctgag 1200 ggctattgag gcgcaacagc atctgttgca actcacagtctggggcatca agcagctcca 1260 ggcaagaatc ctggctgtgg aaagatacct aaaggatcaacagctcctgg ggatttgggg 1320 ttgctctgga aaactcattt gcaccactgc tgtgccttggaatgctagtt ggagtaataa 1380 atctctggaa cagatttgga atcacacgac ctggatggagtgggacagag aaattaacaa 1440 ttacacaagc ttaatacact ccttaattga agaatcgcaaaaccagcaag aaaagaatga 1500 acaagaatta ttggaattag ataaatgggc aagtttgtggaattggttta acataacaaa 1560 ttggctgtgg tatataaaat tattcataat gatagtaggaggcttggtag gtttaagaat 1620 agtttttgct gtactttcta tagtgaatag agttaggcagggatattcac cattatcgtt 1680 tcagacccac ctcccaaccc cgaggggacc cgacaggcccgaaggaatag aagaagaagg 1740 tggagagaga gacagagaca gatccattcg attagtgaacggatctcgac ggtatcgata 1800 agcttgggag ttccgcgtta cataacttac ggtaaatggcccgcctggct gaccgcccaa 1860 cgacccccgc ccattgacgt caataatgac gtatgttcccatagtaacgc caatagggac 1920 tttccattga cgtcaatggg tggagtattt acggtaaactgcccacttgg cagtacatca 1980 agtgtatcat atgccaagta cgccccctat tgacgtcaatgacggtaaat ggcccgcctg 2040 gcattatgcc cagtacatga ccttatggga ctttcctacttggcagtaca tctacgtatt 2100 agtcatcgct attaccatgg tgatgcggtt ttggcagtacatcaatgggc gtggatagcg 2160 gtttgactca cggggatttc caagtctcca ccccattgacgtcaatggga gtttgttttg 2220 gcaccaaaat caacgggact ttccaaaatg tcgtaacaactccgccccat tgacgcaaat 2280 gggcggtagg cgtgtacggt gggaggtcta tataagcagagctcgtttag tgaaccgtca 2340 gatcgcctgg agacgccatc cacgctgttt tgacctccatagaagacacc gactctagag 2400 gatccactag tccagtgtgg tggaattctg cagatatcaacaagtttgta caaaaaagct 2460 gaacgagaaa cgtaaaatga tataaatatc aatatattaaattagatttt gcataaaaaa 2520 cagactacat aatactgtaa aacacaacat atccagtcactatggcggcc gcattaggca 2580 ccccaggctt tacactttat gcttccggct cgtataatgtgtggattttg agttaggatc 2640 cggcgagatt ttcaggagct aaggaagcta aaatggagaaaaaaatcact ggatatacca 2700 ccgttgatat atcccaatgg catcgtaaag aacattttgaggcatttcag tcagttgctc 2760 aatgtaccta taaccagacc gttcagctgg atattacggcctttttaaag accgtaaaga 2820 aaaataagca caagttttat ccggccttta ttcacattcttgcccgcctg atgaatgctc 2880 atccggaatt ccgtatggca atgaaagacg gtgagctggtgatatgggat agtgttcacc 2940 cttgttacac cgttttccat gagcaaactg aaacgttttcatcgctctgg agtgaatacc 3000 acgacgattt ccggcagttt ctacacatat attcgcaagatgtggcgtgt tacggtgaaa 3060 acctggccta tttccctaaa gggtttattg agaatatgtttttcgtctca gccaatccct 3120 gggtgagttt caccagtttt gatttaaacg tggccaatatggacaacttc ttcgcccccg 3180 ttttcaccat gggcaaatat tatacgcaag gcgacaaggtgctgatgccg ctggcgattc 3240 aggttcatca tgccgtctgt gatggcttcc atgtcggcagaatgcttaat gaattacaac 3300 agtactgcga tgagtggcag ggcggggcgt aaagatctggatccggctta ctaaaagcca 3360 gataacagta tgcgtatttg cgcgctgatt tttgcggtataagaatatat actgatatgt 3420 atacccgaag tatgtcaaaa agaggtgtgc tatgaagcagcgtattacag tgacagttga 3480 cagcgacagc tatcagttgc tcaaggcata tatgatgtcaatatctccgg tctggtaagc 3540 acaaccatgc agaatgaagc ccgtcgtctg cgtgccgaacgctggaaagc ggaaaatcag 3600 gaagggatgg ctgaggtcgc ccggtttatt gaaatgaacggctcttttgc tgacgagaac 3660 agggactggt gaaatgcagt ttaaggttta cacctataaaagagagagcc gttatcgtct 3720 gtttgtggat gtacagagtg atattattga cacgcccgggcgacggatgg tgatccccct 3780 ggccagtgca cgtctgctgt cagataaagt ctcccgtgaactttacccgg tggtgcatat 3840 cggggatgaa agctggcgca tgatgaccac cgatatggccagtgtgccgg tctccgttat 3900 cggggaagaa gtggctgatc tcagccaccg cgaaaatgacatcaaaaacg ccattaacct 3960 gatgttctgg ggaatataaa tgtcaggctc cgttatacacagccagtctg caggtcgacc 4020 atagtgactg gatatgttgt gttttacagt attatgtagtctgtttttta tgcaaaatct 4080 aatttaatat attgatattt atatcatttt acgtttctcgttcagctttc ttgtacaaag 4140 tggttgatat ccagcacagt ggcggccgct cgagtctagagggcccgcgg ttcgaaggta 4200 agcctatccc taaccctctc ctcggtctcg attctacgcgtaccggttag taatgagttt 4260 ggaattaatt ctgtggaatg tgtgtcagtt agggtgtggaaagtccccag gctccccagg 4320 caggcagaag tatgcaaagc atgcatctca attagtcagcaaccaggtgt ggaaagtccc 4380 caggctcccc agcaggcaga agtatgcaaa gcatgcatctcaattagtca gcaaccatag 4440 tcccgcccct aactccgccc atcccgcccc taactccgcccagttccgcc cattctccgc 4500 cccatggctg actaattttt tttatttatg cagaggccgaggccgcctct gcctctgagc 4560 tattccagaa gtagtgagga ggcttttttg gaggcctaggcttttgcaaa aagctcccgg 4620 gagcttgtat atccattttc ggatctgatc agcacgtgttgacaattaat catcggcata 4680 gtatatcggc atagtataat acgacaaggt gaggaactaaaccatggcca agcctttgtc 4740 tcaagaagaa tccaccctca ttgaaagagc aacggctacaatcaacagca tccccatctc 4800 tgaagactac agcgtcgcca gcgcagctct ctctagcgacggccgcatct tcactggtgt 4860 caatgtatat cattttactg ggggaccttg tgcagaactcgtggtgctgg gcactgctgc 4920 tgctgcggca gctggcaacc tgacttgtat cgtcgcgatcggaaatgaga acaggggcat 4980 cttgagcccc tgcggacggt gccgacaggt gcttctcgatctgcatcctg ggatcaaagc 5040 catagtgaag gacagtgatg gacagccgac ggcagttgggattcgtgaat tgctgccctc 5100 tggttatgtg tgggagggct aagcacaatt cgagctcggtacctttaaga ccaatgactt 5160 acaaggcagc tgtagatctt agccactttt taaaagaaaaggggggactg gaagggctaa 5220 ttcactccca acgaagacaa gatctgcttt ttgcttgtactgggtctctc tggttagacc 5280 agatctgagc ctgggagctc tctggctaac tagggaacccactgcttaag cctcaataaa 5340 gcttgccttg agtgcttcaa gtagtgtgtg cccgtctgttgtgtgactct ggtaactaga 5400 gatccctcag acccttttag tcagtgtgga aaatctctagcagtagtagt tcatgtcatc 5460 ttattattca gtatttataa cttgcaaaga aatgaatatcagagagtgag aggaacttgt 5520 ttattgcagc ttataatggt tacaaataaa gcaatagcatcacaaatttc acaaataaag 5580 catttttttc actgcattct agttgtggtt tgtccaaactcatcaatgta tcttatcatg 5640 tctggctcta gctatcccgc ccctaactcc gcccatcccgcccctaactc cgcccagttc 5700 cgcccattct ccgccccatg gctgactaat tttttttatttatgcagagg ccgaggccgc 5760 ctcggcctct gagctattcc agaagtagtg aggaggcttttttggaggcc tagggacgta 5820 cccaattcgc cctatagtga gtcgtattac gcgcgctcactggccgtcgt tttacaacgt 5880 cgtgactggg aaaaccctgg cgttacccaa cttaatcgccttgcagcaca tccccctttc 5940 gccagctggc gtaatagcga agaggcccgc accgatcgcccttcccaaca gttgcgcagc 6000 ctgaatggcg aatgggacgc gccctgtagc ggcgcattaagcgcggcggg tgtggtggtt 6060 acgcgcagcg tgaccgctac acttgccagc gccctagcgcccgctccttt cgctttcttc 6120 ccttcctttc tcgccacgtt cgccggcttt ccccgtcaagctctaaatcg ggggctccct 6180 ttagggttcc gatttagtgc tttacggcac ctcgaccccaaaaaacttga ttagggtgat 6240 ggttcacgta gtgggccatc gccctgatag acggtttttcgccctttgac gttggagtcc 6300 acgttcttta atagtggact cttgttccaa actggaacaacactcaaccc tatctcggtc 6360 tattcttttg atttataagg gattttgccg atttcggcctattggttaaa aaatgagctg 6420 atttaacaaa aatttaacgc gaattttaac aaaatattaacgcttacaat ttaggtggca 6480 cttttcgggg aaatgtgcgc ggaaccccta tttgtttatttttctaaata cattcaaata 6540 tgtatccgct catgagacaa taaccctgat aaatgcttcaataatattga aaaaggaaga 6600 gtatgagtat tcaacatttc cgtgtcgccc ttattcccttttttgcggca ttttgccttc 6660 ctgtttttgc tcacccagaa acgctggtga aagtaaaagatgctgaagat cagttgggtg 6720 cacgagtggg ttacatcgaa ctggatctca acagcggtaagatccttgag agttttcgcc 6780 ccgaagaacg ttttccaatg atgagcactt ttaaagttctgctatgtggc gcggtattat 6840 cccgtattga cgccgggcaa gagcaactcg gtcgccgcatacactattct cagaatgact 6900 tggttgagta ctcaccagtc acagaaaagc atcttacggatggcatgaca gtaagagaat 6960 tatgcagtgc tgccataacc atgagtgata acactgcggccaacttactt ctgacaacga 7020 tcggaggacc gaaggagcta accgcttttt tgcacaacatgggggatcat gtaactcgcc 7080 ttgatcgttg ggaaccggag ctgaatgaag ccataccaaacgacgagcgt gacaccacga 7140 tgcctgtagc aatggcaaca acgttgcgca aactattaactggcgaacta cttactctag 7200 cttcccggca acaattaata gactggatgg aggcggataaagttgcagga ccacttctgc 7260 gctcggccct tccggctggc tggtttattg ctgataaatctggagccggt gagcgtgggt 7320 ctcgcggtat cattgcagca ctggggccag atggtaagccctcccgtatc gtagttatct 7380 acacgacggg gagtcaggca actatggatg aacgaaatagacagatcgct gagataggtg 7440 cctcactgat taagcattgg taactgtcag accaagtttactcatatata ctttagattg 7500 atttaaaact tcatttttaa tttaaaagga tctaggtgaagatccttttt gataatctca 7560 tgaccaaaat cccttaacgt gagttttcgt tccactgagcgtcagacccc gtagaaaaga 7620 tcaaaggatc ttcttgagat cctttttttc tgcgcgtaatctgctgcttg caaacaaaaa 7680 aaccaccgct accagcggtg gtttgtttgc cggatcaagagctaccaact ctttttccga 7740 aggtaactgg cttcagcaga gcgcagatac caaatactgttcttctagtg tagccgtagt 7800 taggccacca cttcaagaac tctgtagcac cgcctacatacctcgctctg ctaatcctgt 7860 taccagtggc tgctgccagt ggcgataagt cgtgtcttaccgggttggac tcaagacgat 7920 agttaccgga taaggcgcag cggtcgggct gaacggggggttcgtgcaca cagcccagct 7980 tggagcgaac gacctacacc gaactgagat acctacagcgtgagctatga gaaagcgcca 8040 cgcttcccga agggagaaag gcggacaggt atccggtaagcggcagggtc ggaacaggag 8100 agcgcacgag ggagcttcca gggggaaacg cctggtatctttatagtcct gtcgggtttc 8160 gccacctctg acttgagcgt cgatttttgt gatgctcgtcaggggggcgg agcctatgga 8220 aaaacgccag caacgcggcc tttttacggt tcctggccttttgctggcct tttgctcaca 8280 tgttctttcc tgcgttatcc cctgattctg tggataaccgtattaccgcc tttgagtgag 8340 ctgataccgc tcgccgcagc cgaacgaccg agcgcagcgagtcagtgagc gaggaagcgg 8400 aagagcgccc aatacgcaaa ccgcctctcc ccgcgcgttggccgattcat taatgcagct 8460 ggcacgacag gtttcccgac tggaaagcgg gcagtgagcgcaacgcaatt aatgtgagtt 8520 agctcactca ttaggcaccc caggctttac actttatgcttccggctcgt atgttgtgtg 8580 gaattgtgag cggataacaa tttcacacag gaaacagctatgaccatgat tacgccaagc 8640 gcgcaattaa ccctcactaa agggaacaaa agctggagctgcaagctt 8688 106 6964 DNA Artificial Sequence pLenti6/V5-D-TOPO 106aatgtagtct tatgcaatac tcttgtagtc ttgcaacatg gtaacgatga gttagcaaca 60tgccttacaa ggagagaaaa agcaccgtgc atgccgattg gtggaagtaa ggtggtacga 120tcgtgcctta ttaggaaggc aacagacggg tctgacatgg attggacgaa ccactgaatt 180gccgcattgc agagatattg tatttaagtg cctagctcga tacataaacg ggtctctctg 240gttagaccag atctgagcct gggagctctc tggctaacta gggaacccac tgcttaagcc 300tcaataaagc ttgccttgag tgcttcaagt agtgtgtgcc cgtctgttgt gtgactctgg 360taactagaga tccctcagac ccttttagtc agtgtggaaa atctctagca gtggcgcccg 420aacagggact tgaaagcgaa agggaaacca gaggagctct ctcgacgcag gactcggctt 480gctgaagcgc gcacggcaag aggcgagggg cggcgactgg tgagtacgcc aaaaattttg 540actagcggag gctagaagga gagagatggg tgcgagagcg tcagtattaa gcgggggaga 600attagatcgc gatgggaaaa aattcggtta aggccagggg gaaagaaaaa atataaatta 660aaacatatag tatgggcaag cagggagcta gaacgattcg cagttaatcc tggcctgtta 720gaaacatcag aaggctgtag acaaatactg ggacagctac aaccatccct tcagacagga 780tcagaagaac ttagatcatt atataataca gtagcaaccc tctattgtgt gcatcaaagg 840atagagataa aagacaccaa ggaagcttta gacaagatag aggaagagca aaacaaaagt 900aagaccaccg cacagcaagc ggccgctgat cttcagacct ggaggaggag atatgaggga 960caattggaga agtgaattat ataaatataa agtagtaaaa attgaaccat taggagtagc 1020acccaccaag gcaaagagaa gagtggtgca gagagaaaaa agagcagtgg gaataggagc 1080tttgttcctt gggttcttgg gagcagcagg aagcactatg ggcgcagcgt caatgacgct 1140gacggtacag gccagacaat tattgtctgg tatagtgcag cagcagaaca atttgctgag 1200ggctattgag gcgcaacagc atctgttgca actcacagtc tggggcatca agcagctcca 1260ggcaagaatc ctggctgtgg aaagatacct aaaggatcaa cagctcctgg ggatttgggg 1320ttgctctgga aaactcattt gcaccactgc tgtgccttgg aatgctagtt ggagtaataa 1380atctctggaa cagatttgga atcacacgac ctggatggag tgggacagag aaattaacaa 1440ttacacaagc ttaatacact ccttaattga agaatcgcaa aaccagcaag aaaagaatga 1500acaagaatta ttggaattag ataaatgggc aagtttgtgg aattggttta acataacaaa 1560ttggctgtgg tatataaaat tattcataat gatagtagga ggcttggtag gtttaagaat 1620agtttttgct gtactttcta tagtgaatag agttaggcag ggatattcac cattatcgtt 1680tcagacccac ctcccaaccc cgaggggacc cgacaggccc gaaggaatag aagaagaagg 1740tggagagaga gacagagaca gatccattcg attagtgaac ggatctcgac ggtatcgata 1800agcttgggag ttccgcgtta cataacttac ggtaaatggc ccgcctggct gaccgcccaa 1860cgacccccgc ccattgacgt caataatgac gtatgttccc atagtaacgc caatagggac 1920tttccattga cgtcaatggg tggagtattt acggtaaact gcccacttgg cagtacatca 1980agtgtatcat atgccaagta cgccccctat tgacgtcaat gacggtaaat ggcccgcctg 2040gcattatgcc cagtacatga ccttatggga ctttcctact tggcagtaca tctacgtatt 2100agtcatcgct attaccatgg tgatgcggtt ttggcagtac atcaatgggc gtggatagcg 2160gtttgactca cggggatttc caagtctcca ccccattgac gtcaatggga gtttgttttg 2220gcaccaaaat caacgggact ttccaaaatg tcgtaacaac tccgccccat tgacgcaaat 2280gggcggtagg cgtgtacggt gggaggtcta tataagcaga gctcgtttag tgaaccgtca 2340gatcgcctgg agacgccatc cacgctgttt tgacctccat agaagacacc gactctagag 2400gatccactag tccagtgtgg tggaattgat cccttcacca agggctcgag tctagagggc 2460ccgcggttcg aaggtaagcc tatccctaac cctctcctcg gtctcgattc tacgcgtacc 2520ggttagtaat gagtttggaa ttaattctgt ggaatgtgtg tcagttaggg tgtggaaagt 2580ccccaggctc cccaggcagg cagaagtatg caaagcatgc atctcaatta gtcagcaacc 2640aggtgtggaa agtccccagg ctccccagca ggcagaagta tgcaaagcat gcatctcaat 2700tagtcagcaa ccatagtccc gcccctaact ccgcccatcc cgcccctaac tccgcccagt 2760tccgcccatt ctccgcccca tggctgacta atttttttta tttatgcaga ggccgaggcc 2820gcctctgcct ctgagctatt ccagaagtag tgaggaggct tttttggagg cctaggcttt 2880tgcaaaaagc tcccgggagc ttgtatatcc attttcggat ctgatcagca cgtgttgaca 2940attaatcatc ggcatagtat atcggcatag tataatacga caaggtgagg aactaaacca 3000tggccaagcc tttgtctcaa gaagaatcca ccctcattga aagagcaacg gctacaatca 3060acagcatccc catctctgaa gactacagcg tcgccagcgc agctctctct agcgacggcc 3120gcatcttcac tggtgtcaat gtatatcatt ttactggggg accttgtgca gaactcgtgg 3180tgctgggcac tgctgctgct gcggcagctg gcaacctgac ttgtatcgtc gcgatcggaa 3240atgagaacag gggcatcttg agcccctgcg gacggtgccg acaggtgctt ctcgatctgc 3300atcctgggat caaagccata gtgaaggaca gtgatggaca gccgacggca gttgggattc 3360gtgaattgct gccctctggt tatgtgtggg agggctaagc acaattcgag ctcggtacct 3420ttaagaccaa tgacttacaa ggcagctgta gatcttagcc actttttaaa agaaaagggg 3480ggactggaag ggctaattca ctcccaacga agacaagatc tgctttttgc ttgtactggg 3540tctctctggt tagaccagat ctgagcctgg gagctctctg gctaactagg gaacccactg 3600cttaagcctc aataaagctt gccttgagtg cttcaagtag tgtgtgcccg tctgttgtgt 3660gactctggta actagagatc cctcagaccc ttttagtcag tgtggaaaat ctctagcagt 3720agtagttcat gtcatcttat tattcagtat ttataacttg caaagaaatg aatatcagag 3780agtgagagga acttgtttat tgcagcttat aatggttaca aataaagcaa tagcatcaca 3840aatttcacaa ataaagcatt tttttcactg cattctagtt gtggtttgtc caaactcatc 3900aatgtatctt atcatgtctg gctctagcta tcccgcccct aactccgccc atcccgcccc 3960taactccgcc cagttccgcc cattctccgc cccatggctg actaattttt tttatttatg 4020cagaggccga ggccgcctcg gcctctgagc tattccagaa gtagtgagga ggcttttttg 4080gaggcctagg gacgtaccca attcgcccta tagtgagtcg tattacgcgc gctcactggc 4140cgtcgtttta caacgtcgtg actgggaaaa ccctggcgtt acccaactta atcgccttgc 4200agcacatccc cctttcgcca gctggcgtaa tagcgaagag gcccgcaccg atcgcccttc 4260ccaacagttg cgcagcctga atggcgaatg ggacgcgccc tgtagcggcg cattaagcgc 4320ggcgggtgtg gtggttacgc gcagcgtgac cgctacactt gccagcgccc tagcgcccgc 4380tcctttcgct ttcttccctt cctttctcgc cacgttcgcc ggctttcccc gtcaagctct 4440aaatcggggg ctccctttag ggttccgatt tagtgcttta cggcacctcg accccaaaaa 4500acttgattag ggtgatggtt cacgtagtgg gccatcgccc tgatagacgg tttttcgccc 4560tttgacgttg gagtccacgt tctttaatag tggactcttg ttccaaactg gaacaacact 4620caaccctatc tcggtctatt cttttgattt ataagggatt ttgccgattt cggcctattg 4680gttaaaaaat gagctgattt aacaaaaatt taacgcgaat tttaacaaaa tattaacgct 4740tacaatttag gtggcacttt tcggggaaat gtgcgcggaa cccctatttg tttatttttc 4800taaatacatt caaatatgta tccgctcatg agacaataac cctgataaat gcttcaataa 4860tattgaaaaa ggaagagtat gagtattcaa catttccgtg tcgcccttat tccctttttt 4920gcggcatttt gccttcctgt ttttgctcac ccagaaacgc tggtgaaagt aaaagatgct 4980gaagatcagt tgggtgcacg agtgggttac atcgaactgg atctcaacag cggtaagatc 5040cttgagagtt ttcgccccga agaacgtttt ccaatgatga gcacttttaa agttctgcta 5100tgtggcgcgg tattatcccg tattgacgcc gggcaagagc aactcggtcg ccgcatacac 5160tattctcaga atgacttggt tgagtactca ccagtcacag aaaagcatct tacggatggc 5220atgacagtaa gagaattatg cagtgctgcc ataaccatga gtgataacac tgcggccaac 5280ttacttctga caacgatcgg aggaccgaag gagctaaccg cttttttgca caacatgggg 5340gatcatgtaa ctcgccttga tcgttgggaa ccggagctga atgaagccat accaaacgac 5400gagcgtgaca ccacgatgcc tgtagcaatg gcaacaacgt tgcgcaaact attaactggc 5460gaactactta ctctagcttc ccggcaacaa ttaatagact ggatggaggc ggataaagtt 5520gcaggaccac ttctgcgctc ggcccttccg gctggctggt ttattgctga taaatctgga 5580gccggtgagc gtgggtctcg cggtatcatt gcagcactgg ggccagatgg taagccctcc 5640cgtatcgtag ttatctacac gacggggagt caggcaacta tggatgaacg aaatagacag 5700atcgctgaga taggtgcctc actgattaag cattggtaac tgtcagacca agtttactca 5760tatatacttt agattgattt aaaacttcat ttttaattta aaaggatcta ggtgaagatc 5820ctttttgata atctcatgac caaaatccct taacgtgagt tttcgttcca ctgagcgtca 5880gaccccgtag aaaagatcaa aggatcttct tgagatcctt tttttctgcg cgtaatctgc 5940tgcttgcaaa caaaaaaacc accgctacca gcggtggttt gtttgccgga tcaagagcta 6000ccaactcttt ttccgaaggt aactggcttc agcagagcgc agataccaaa tactgttctt 6060ctagtgtagc cgtagttagg ccaccacttc aagaactctg tagcaccgcc tacatacctc 6120gctctgctaa tcctgttacc agtggctgct gccagtggcg ataagtcgtg tcttaccggg 6180ttggactcaa gacgatagtt accggataag gcgcagcggt cgggctgaac ggggggttcg 6240tgcacacagc ccagcttgga gcgaacgacc tacaccgaac tgagatacct acagcgtgag 6300ctatgagaaa gcgccacgct tcccgaaggg agaaaggcgg acaggtatcc ggtaagcggc 6360agggtcggaa caggagagcg cacgagggag cttccagggg gaaacgcctg gtatctttat 6420agtcctgtcg ggtttcgcca cctctgactt gagcgtcgat ttttgtgatg ctcgtcaggg 6480gggcggagcc tatggaaaaa cgccagcaac gcggcctttt tacggttcct ggccttttgc 6540tggccttttg ctcacatgtt ctttcctgcg ttatcccctg attctgtgga taaccgtatt 6600accgcctttg agtgagctga taccgctcgc cgcagccgaa cgaccgagcg cagcgagtca 6660gtgagcgagg aagcggaaga gcgcccaata cgcaaaccgc ctctccccgc gcgttggccg 6720attcattaat gcagctggca cgacaggttt cccgactgga aagcgggcag tgagcgcaac 6780gcaattaatg tgagttagct cactcattag gcaccccagg ctttacactt tatgcttccg 6840gctcgtatgt tgtgtggaat tgtgagcgga taacaatttc acacaggaaa cagctatgac 6900catgattacg ccaagcgcgc aattaaccct cactaaaggg aacaaaagct ggagctgcaa 6960gctt 6964 107 8634 DNA Artificial Sequence pLenti4/V5-DEST 107aatgtagtct tatgcaatac tcttgtagtc ttgcaacatg gtaacgatga gttagcaaca 60tgccttacaa ggagagaaaa agcaccgtgc atgccgattg gtggaagtaa ggtggtacga 120tcgtgcctta ttaggaaggc aacagacggg tctgacatgg attggacgaa ccactgaatt 180gccgcattgc agagatattg tatttaagtg cctagctcga tacataaacg ggtctctctg 240gttagaccag atctgagcct gggagctctc tggctaacta gggaacccac tgcttaagcc 300tcaataaagc ttgccttgag tgcttcaagt agtgtgtgcc cgtctgttgt gtgactctgg 360taactagaga tccctcagac ccttttagtc agtgtggaaa atctctagca gtggcgcccg 420aacagggact tgaaagcgaa agggaaacca gaggagctct ctcgacgcag gactcggctt 480gctgaagcgc gcacggcaag aggcgagggg cggcgactgg tgagtacgcc aaaaattttg 540actagcggag gctagaagga gagagatggg tgcgagagcg tcagtattaa gcgggggaga 600attagatcgc gatgggaaaa aattcggtta aggccagggg gaaagaaaaa atataaatta 660aaacatatag tatgggcaag cagggagcta gaacgattcg cagttaatcc tggcctgtta 720gaaacatcag aaggctgtag acaaatactg ggacagctac aaccatccct tcagacagga 780tcagaagaac ttagatcatt atataataca gtagcaaccc tctattgtgt gcatcaaagg 840atagagataa aagacaccaa ggaagcttta gacaagatag aggaagagca aaacaaaagt 900aagaccaccg cacagcaagc ggccgctgat cttcagacct ggaggaggag atatgaggga 960caattggaga agtgaattat ataaatataa agtagtaaaa attgaaccat taggagtagc 1020acccaccaag gcaaagagaa gagtggtgca gagagaaaaa agagcagtgg gaataggagc 1080tttgttcctt gggttcttgg gagcagcagg aagcactatg ggcgcagcgt caatgacgct 1140gacggtacag gccagacaat tattgtctgg tatagtgcag cagcagaaca atttgctgag 1200ggctattgag gcgcaacagc atctgttgca actcacagtc tggggcatca agcagctcca 1260ggcaagaatc ctggctgtgg aaagatacct aaaggatcaa cagctcctgg ggatttgggg 1320ttgctctgga aaactcattt gcaccactgc tgtgccttgg aatgctagtt ggagtaataa 1380atctctggaa cagatttgga atcacacgac ctggatggag tgggacagag aaattaacaa 1440ttacacaagc ttaatacact ccttaattga agaatcgcaa aaccagcaag aaaagaatga 1500acaagaatta ttggaattag ataaatgggc aagtttgtgg aattggttta acataacaaa 1560ttggctgtgg tatataaaat tattcataat gatagtagga ggcttggtag gtttaagaat 1620agtttttgct gtactttcta tagtgaatag agttaggcag ggatattcac cattatcgtt 1680tcagacccac ctcccaaccc cgaggggacc cgacaggccc gaaggaatag aagaagaagg 1740tggagagaga gacagagaca gatccattcg attagtgaac ggatctcgac ggtatcgata 1800agcttgggag ttccgcgtta cataacttac ggtaaatggc ccgcctggct gaccgcccaa 1860cgacccccgc ccattgacgt caataatgac gtatgttccc atagtaacgc caatagggac 1920tttccattga cgtcaatggg tggagtattt acggtaaact gcccacttgg cagtacatca 1980agtgtatcat atgccaagta cgccccctat tgacgtcaat gacggtaaat ggcccgcctg 2040gcattatgcc cagtacatga ccttatggga ctttcctact tggcagtaca tctacgtatt 2100agtcatcgct attaccatgg tgatgcggtt ttggcagtac atcaatgggc gtggatagcg 2160gtttgactca cggggatttc caagtctcca ccccattgac gtcaatggga gtttgttttg 2220gcaccaaaat caacgggact ttccaaaatg tcgtaacaac tccgccccat tgacgcaaat 2280gggcggtagg cgtgtacggt gggaggtcta tataagcaga gctcgtttag tgaaccgtca 2340gatcgcctgg agacgccatc cacgctgttt tgacctccat agaagacacc gactctagag 2400gatccactag tccagtgtgg tggaattctg cagatatcaa caagtttgta caaaaaagct 2460gaacgagaaa cgtaaaatga tataaatatc aatatattaa attagatttt gcataaaaaa 2520cagactacat aatactgtaa aacacaacat atccagtcac tatggcggcc gcattaggca 2580ccccaggctt tacactttat gcttccggct cgtataatgt gtggattttg agttaggatc 2640cggcgagatt ttcaggagct aaggaagcta aaatggagaa aaaaatcact ggatatacca 2700ccgttgatat atcccaatgg catcgtaaag aacattttga ggcatttcag tcagttgctc 2760aatgtaccta taaccagacc gttcagctgg atattacggc ctttttaaag accgtaaaga 2820aaaataagca caagttttat ccggccttta ttcacattct tgcccgcctg atgaatgctc 2880atccggaatt ccgtatggca atgaaagacg gtgagctggt gatatgggat agtgttcacc 2940cttgttacac cgttttccat gagcaaactg aaacgttttc atcgctctgg agtgaatacc 3000acgacgattt ccggcagttt ctacacatat attcgcaaga tgtggcgtgt tacggtgaaa 3060acctggccta tttccctaaa gggtttattg agaatatgtt tttcgtctca gccaatccct 3120gggtgagttt caccagtttt gatttaaacg tggccaatat ggacaacttc ttcgcccccg 3180ttttcaccat gggcaaatat tatacgcaag gcgacaaggt gctgatgccg ctggcgattc 3240aggttcatca tgccgtctgt gatggcttcc atgtcggcag aatgcttaat gaattacaac 3300agtactgcga tgagtggcag ggcggggcgt aaagatctgg atccggctta ctaaaagcca 3360gataacagta tgcgtatttg cgcgctgatt tttgcggtat aagaatatat actgatatgt 3420atacccgaag tatgtcaaaa agaggtgtgc tatgaagcag cgtattacag tgacagttga 3480cagcgacagc tatcagttgc tcaaggcata tatgatgtca atatctccgg tctggtaagc 3540acaaccatgc agaatgaagc ccgtcgtctg cgtgccgaac gctggaaagc ggaaaatcag 3600gaagggatgg ctgaggtcgc ccggtttatt gaaatgaacg gctcttttgc tgacgagaac 3660agggactggt gaaatgcagt ttaaggttta cacctataaa agagagagcc gttatcgtct 3720gtttgtggat gtacagagtg atattattga cacgcccggg cgacggatgg tgatccccct 3780ggccagtgca cgtctgctgt cagataaagt ctcccgtgaa ctttacccgg tggtgcatat 3840cggggatgaa agctggcgca tgatgaccac cgatatggcc agtgtgccgg tctccgttat 3900cggggaagaa gtggctgatc tcagccaccg cgaaaatgac atcaaaaacg ccattaacct 3960gatgttctgg ggaatataaa tgtcaggctc cgttatacac agccagtctg caggtcgacc 4020atagtgactg gatatgttgt gttttacagt attatgtagt ctgtttttta tgcaaaatct 4080aatttaatat attgatattt atatcatttt acgtttctcg ttcagctttc ttgtacaaag 4140tggttgatat ccagcacagt ggcggccgct cgagtctaga gggcccgcgg ttcgaaggta 4200agcctatccc taaccctctc ctcggtctcg attctacgcg taccggttag taatgagttt 4260ggaattaatt ctgtggaatg tgtgtcagtt agggtgtgga aagtccccag gctccccagg 4320caggcagaag tatgcaaagc atgcatctca attagtcagc aaccaggtgt ggaaagtccc 4380caggctcccc agcaggcaga agtatgcaaa gcatgcatct caattagtca gcaaccatag 4440tcccgcccct aactccgccc atcccgcccc taactccgcc cagttccgcc cattctccgc 4500cccatggctg actaattttt tttatttatg cagaggccga ggccgcctct gcctctgagc 4560tattccagaa gtagtgagga ggcttttttg gaggcctagg cttttgcaaa aagctccccc 4620tgttgacaat taatcatcgg catagtatat cggcatagta taatacgaca aggtgaggaa 4680ctaaaccatg gccaagttga ccagtgccgt tccggtgctc accgcgcgcg acgtcgccgg 4740agcggtcgag ttctggaccg accggctcgg gttctcccgg gacttcgtgg aggacgactt 4800cgccggtgtg gtccgggacg acgtgaccct gttcatcagc gcggtccagg accaggtggt 4860gccggacaac accctggcct gggtgtgggt gcgcggcctg gacgagctgt acgccgagtg 4920gtcggaggtc gtgtccacga acttccggga cgcctccggg ccggccatga ccgagatcgg 4980cgagcagccg tgggggcggg agttcgccct gcgcgacccg gccggcaact gcgtgcactt 5040cgtggccgag gagcaggact gacacgtgct acgagattta aatggtacct ttaagaccaa 5100tgacttacaa ggcagctgta gatcttagcc actttttaaa agaaaagggg ggactggaag 5160ggctaattca ctcccaacga agacaagatc tgctttttgc ttgtactggg tctctctggt 5220tagaccagat ctgagcctgg gagctctctg gctaactagg gaacccactg cttaagcctc 5280aataaagctt gccttgagtg cttcaagtag tgtgtgcccg tctgttgtgt gactctggta 5340actagagatc cctcagaccc ttttagtcag tgtggaaaat ctctagcagt agtagttcat 5400gtcatcttat tattcagtat ttataacttg caaagaaatg aatatcagag agtgagagga 5460acttgtttat tgcagcttat aatggttaca aataaagcaa tagcatcaca aatttcacaa 5520ataaagcatt tttttcactg cattctagtt gtggtttgtc caaactcatc aatgtatctt 5580atcatgtctg gctctagcta tcccgcccct aactccgccc atcccgcccc taactccgcc 5640cagttccgcc cattctccgc cccatggctg actaattttt tttatttatg cagaggccga 5700ggccgcctcg gcctctgagc tattccagaa gtagtgagga ggcttttttg gaggcctagg 5760gacgtaccca attcgcccta tagtgagtcg tattacgcgc gctcactggc cgtcgtttta 5820caacgtcgtg actgggaaaa ccctggcgtt acccaactta atcgccttgc agcacatccc 5880cctttcgcca gctggcgtaa tagcgaagag gcccgcaccg atcgcccttc ccaacagttg 5940cgcagcctga atggcgaatg ggacgcgccc tgtagcggcg cattaagcgc ggcgggtgtg 6000gtggttacgc gcagcgtgac cgctacactt gccagcgccc tagcgcccgc tcctttcgct 6060ttcttccctt cctttctcgc cacgttcgcc ggctttcccc gtcaagctct aaatcggggg 6120ctccctttag ggttccgatt tagtgcttta cggcacctcg accccaaaaa acttgattag 6180ggtgatggtt cacgtagtgg gccatcgccc tgatagacgg tttttcgccc tttgacgttg 6240gagtccacgt tctttaatag tggactcttg ttccaaactg gaacaacact caaccctatc 6300tcggtctatt cttttgattt ataagggatt ttgccgattt cggcctattg gttaaaaaat 6360gagctgattt aacaaaaatt taacgcgaat tttaacaaaa tattaacgct tacaatttag 6420gtggcacttt tcggggaaat gtgcgcggaa cccctatttg tttatttttc taaatacatt 6480caaatatgta tccgctcatg agacaataac cctgataaat gcttcaataa tattgaaaaa 6540ggaagagtat gagtattcaa catttccgtg tcgcccttat tccctttttt gcggcatttt 6600gccttcctgt ttttgctcac ccagaaacgc tggtgaaagt aaaagatgct gaagatcagt 6660tgggtgcacg agtgggttac atcgaactgg atctcaacag cggtaagatc cttgagagtt 6720ttcgccccga agaacgtttt ccaatgatga gcacttttaa agttctgcta tgtggcgcgg 6780tattatcccg tattgacgcc gggcaagagc aactcggtcg ccgcatacac tattctcaga 6840atgacttggt tgagtactca ccagtcacag aaaagcatct tacggatggc atgacagtaa 6900gagaattatg cagtgctgcc ataaccatga gtgataacac tgcggccaac ttacttctga 6960caacgatcgg aggaccgaag gagctaaccg cttttttgca caacatgggg gatcatgtaa 7020ctcgccttga tcgttgggaa ccggagctga atgaagccat accaaacgac gagcgtgaca 7080ccacgatgcc tgtagcaatg gcaacaacgt tgcgcaaact attaactggc gaactactta 7140ctctagcttc ccggcaacaa ttaatagact ggatggaggc ggataaagtt gcaggaccac 7200ttctgcgctc ggcccttccg gctggctggt ttattgctga taaatctgga gccggtgagc 7260gtgggtctcg cggtatcatt gcagcactgg ggccagatgg taagccctcc cgtatcgtag 7320ttatctacac gacggggagt caggcaacta tggatgaacg aaatagacag atcgctgaga 7380taggtgcctc actgattaag cattggtaac tgtcagacca agtttactca tatatacttt 7440agattgattt aaaacttcat ttttaattta aaaggatcta ggtgaagatc ctttttgata 7500atctcatgac caaaatccct taacgtgagt tttcgttcca ctgagcgtca gaccccgtag 7560aaaagatcaa aggatcttct tgagatcctt tttttctgcg cgtaatctgc tgcttgcaaa 7620caaaaaaacc accgctacca gcggtggttt gtttgccgga tcaagagcta ccaactcttt 7680ttccgaaggt aactggcttc agcagagcgc agataccaaa tactgttctt ctagtgtagc 7740cgtagttagg ccaccacttc aagaactctg tagcaccgcc tacatacctc gctctgctaa 7800tcctgttacc agtggctgct gccagtggcg ataagtcgtg tcttaccggg ttggactcaa 7860gacgatagtt accggataag gcgcagcggt cgggctgaac ggggggttcg tgcacacagc 7920ccagcttgga gcgaacgacc tacaccgaac tgagatacct acagcgtgag ctatgagaaa 7980gcgccacgct tcccgaaggg agaaaggcgg acaggtatcc ggtaagcggc agggtcggaa 8040caggagagcg cacgagggag cttccagggg gaaacgcctg gtatctttat agtcctgtcg 8100ggtttcgcca cctctgactt gagcgtcgat ttttgtgatg ctcgtcaggg gggcggagcc 8160tatggaaaaa cgccagcaac gcggcctttt tacggttcct ggccttttgc tggccttttg 8220ctcacatgtt ctttcctgcg ttatcccctg attctgtgga taaccgtatt accgcctttg 8280agtgagctga taccgctcgc cgcagccgaa cgaccgagcg cagcgagtca gtgagcgagg 8340aagcggaaga gcgcccaata cgcaaaccgc ctctccccgc gcgttggccg attcattaat 8400gcagctggca cgacaggttt cccgactgga aagcgggcag tgagcgcaac gcaattaatg 8460tgagttagct cactcattag gcaccccagg ctttacactt tatgcttccg gctcgtatgt 8520tgtgtggaat tgtgagcgga taacaatttc acacaggaaa cagctatgac catgattacg 8580ccaagcgcgc aattaaccct cactaaaggg aacaaaagct ggagctgcaa gctt 8634 1089320 DNA Artificial Sequence pLenti6/UbC/V5-DEST 108 aatgtagtcttatgcaatac tcttgtagtc ttgcaacatg gtaacgatga gttagcaaca 60 tgccttacaaggagagaaaa agcaccgtgc atgccgattg gtggaagtaa ggtggtacga 120 tcgtgccttattaggaaggc aacagacggg tctgacatgg attggacgaa ccactgaatt 180 gccgcattgcagagatattg tatttaagtg cctagctcga tacataaacg ggtctctctg 240 gttagaccagatctgagcct gggagctctc tggctaacta gggaacccac tgcttaagcc 300 tcaataaagcttgccttgag tgcttcaagt agtgtgtgcc cgtctgttgt gtgactctgg 360 taactagagatccctcagac ccttttagtc agtgtggaaa atctctagca gtggcgcccg 420 aacagggacttgaaagcgaa agggaaacca gaggagctct ctcgacgcag gactcggctt 480 gctgaagcgcgcacggcaag aggcgagggg cggcgactgg tgagtacgcc aaaaattttg 540 actagcggaggctagaagga gagagatggg tgcgagagcg tcagtattaa gcgggggaga 600 attagatcgcgatgggaaaa aattcggtta aggccagggg gaaagaaaaa atataaatta 660 aaacatatagtatgggcaag cagggagcta gaacgattcg cagttaatcc tggcctgtta 720 gaaacatcagaaggctgtag acaaatactg ggacagctac aaccatccct tcagacagga 780 tcagaagaacttagatcatt atataataca gtagcaaccc tctattgtgt gcatcaaagg 840 atagagataaaagacaccaa ggaagcttta gacaagatag aggaagagca aaacaaaagt 900 aagaccaccgcacagcaagc ggccgctgat cttcagacct ggaggaggag atatgaggga 960 caattggagaagtgaattat ataaatataa agtagtaaaa attgaaccat taggagtagc 1020 acccaccaaggcaaagagaa gagtggtgca gagagaaaaa agagcagtgg gaataggagc 1080 tttgttccttgggttcttgg gagcagcagg aagcactatg ggcgcagcgt caatgacgct 1140 gacggtacaggccagacaat tattgtctgg tatagtgcag cagcagaaca atttgctgag 1200 ggctattgaggcgcaacagc atctgttgca actcacagtc tggggcatca agcagctcca 1260 ggcaagaatcctggctgtgg aaagatacct aaaggatcaa cagctcctgg ggatttgggg 1320 ttgctctggaaaactcattt gcaccactgc tgtgccttgg aatgctagtt ggagtaataa 1380 atctctggaacagatttgga atcacacgac ctggatggag tgggacagag aaattaacaa 1440 ttacacaagcttaatacact ccttaattga agaatcgcaa aaccagcaag aaaagaatga 1500 acaagaattattggaattag ataaatgggc aagtttgtgg aattggttta acataacaaa 1560 ttggctgtggtatataaaat tattcataat gatagtagga ggcttggtag gtttaagaat 1620 agtttttgctgtactttcta tagtgaatag agttaggcag ggatattcac cattatcgtt 1680 tcagacccacctcccaaccc cgaggggacc cgacaggccc gaaggaatag aagaagaagg 1740 tggagagagagacagagaca gatccattcg attagtgaac ggatctcgac ggtatcggat 1800 ctggcctccgcgccgggttt tggcgcctcc cgcgggcgcc cccctcctca cggcgagcgc 1860 tgccacgtcagacgaagggc gcaggagcgt cctgatcctt ccgcccggac gctcaggaca 1920 gcggcccgctgctcataaga ctcggcctta gaaccccagt atcagcagaa ggacatttta 1980 ggacgggacttgggtgactc tagggcactg gttttctttc cagagagcgg aacaggcgag 2040 gaaaagtagtcccttctcgg cgattctgcg gagggatctc cgtggggcgg tgaacgccga 2100 tgattatataaggacgcgcc gggtgtggca cagctagttc cgtcgcagcc gggatttggg 2160 tcgcggttcttgtttgtgga tcgctgtgat cgtcacttgg tgagtagcgg gctgctgggc 2220 tggccggggctttcgtggcc gccgggccgc tcggtgggac ggaagcgtgt ggagagaccg 2280 ccaagggctgtagtctgggt ccgcgagcaa ggttgccctg aactgggggt tggggggagc 2340 gcagcaaaatggcggctgtt cccgagtctt gaatggaaga cgcttgtgag gcgggctgtg 2400 aggtcgttgaaacaaggtgg ggggcatggt gggcggcaag aacccaaggt cttgaggcct 2460 tcgctaatgcgggaaagctc ttattcgggt gagatgggct ggggcaccat ctggggaccc 2520 tgacgtgaagtttgtcactg actggagaac tcggtttgtc gtctgttgcg ggggcggcag 2580 ttatgcggtgccgttgggca gtgcacccgt acctttggga gcgcgcgccc tcgtcgtgtc 2640 gtgacgtcacccgttctgtt ggcttataat gcagggtggg gccacctgcc ggtaggtgtg 2700 cggtaggcttttctccgtcg caggacgcag ggttcgggcc tagggtaggc tctcctgaat 2760 cgacaggcgccggacctctg gtgaggggag ggataagtga ggcgtcagtt tctttggtcg 2820 gttttatgtacctatcttct taagtagctg aagctccggt tttgaactat gcgctcgggg 2880 ttggcgagtgtgttttgtga agttttttag gcaccttttg aaatgtaatc atttgggtca 2940 atatgtaattttcagtgtta gactagtaaa ttgtccgcta aattctggcc gtttttggct 3000 tttttgttagacgaagcttg gtaccgagct cggatccact agtccagtgt ggtggaattc 3060 tgcagatatcaacaagtttg tacaaaaaag ctgaacgaga aacgtaaaat gatataaata 3120 tcaatatattaaattagatt ttgcataaaa aacagactac ataatactgt aaaacacaac 3180 atatccagtcactatggcgg ccgcattagg caccccaggc tttacacttt atgcttccgg 3240 ctcgtataatgtgtggattt tgagttagga tccggcgaga ttttcaggag ctaaggaagc 3300 taaaatggagaaaaaaatca ctggatatac caccgttgat atatcccaat ggcatcgtaa 3360 agaacattttgaggcatttc agtcagttgc tcaatgtacc tataaccaga ccgttcagct 3420 ggatattacggcctttttaa agaccgtaaa gaaaaataag cacaagtttt atccggcctt 3480 tattcacattcttgcccgcc tgatgaatgc tcatccggaa ttccgtatgg caatgaaaga 3540 cggtgagctggtgatatggg atagtgttca cccttgttac accgttttcc atgagcaaac 3600 tgaaacgttttcatcgctct ggagtgaata ccacgacgat ttccggcagt ttctacacat 3660 atattcgcaagatgtggcgt gttacggtga aaacctggcc tatttcccta aagggtttat 3720 tgagaatatgtttttcgtct cagccaatcc ctgggtgagt ttcaccagtt ttgatttaaa 3780 cgtggccaatatggacaact tcttcgcccc cgttttcacc atgggcaaat attatacgca 3840 aggcgacaaggtgctgatgc cgctggcgat tcaggttcat catgccgtct gtgatggctt 3900 ccatgtcggcagaatgctta atgaattaca acagtactgc gatgagtggc agggcggggc 3960 gtaaagatctggatccggct tactaaaagc cagataacag tatgcgtatt tgcgcgctga 4020 tttttgcggtataagaatat atactgatat gtatacccga agtatgtcaa aaagaggtgt 4080 gctatgaagcagcgtattac agtgacagtt gacagcgaca gctatcagtt gctcaaggca 4140 tatatgatgtcaatatctcc ggtctggtaa gcacaaccat gcagaatgaa gcccgtcgtc 4200 tgcgtgccgaacgctggaaa gcggaaaatc aggaagggat ggctgaggtc gcccggttta 4260 ttgaaatgaacggctctttt gctgacgaga acagggactg gtgaaatgca gtttaaggtt 4320 tacacctataaaagagagag ccgttatcgt ctgtttgtgg atgtacagag tgatattatt 4380 gacacgcccgggcgacggat ggtgatcccc ctggccagtg cacgtctgct gtcagataaa 4440 gtctcccgtgaactttaccc ggtggtgcat atcggggatg aaagctggcg catgatgacc 4500 accgatatggccagtgtgcc ggtctccgtt atcggggaag aagtggctga tctcagccac 4560 cgcgaaaatgacatcaaaaa cgccattaac ctgatgttct ggggaatata aatgtcaggc 4620 tccgttatacacagccagtc tgcaggtcga ccatagtgac tggatatgtt gtgttttaca 4680 gtattatgtagtctgttttt tatgcaaaat ctaatttaat atattgatat ttatatcatt 4740 ttacgtttctcgttcagctt tcttgtacaa agtggttgat atccagcaca gtggcggccg 4800 ctcgagtctagagggcccgc ggttcgaagg taagcctatc cctaaccctc tcctcggtct 4860 cgattctacgcgtaccggtt agtaatgagt ttggaattaa ttctgtggaa tgtgtgtcag 4920 ttagggtgtggaaagtcccc aggctcccca ggcaggcaga agtatgcaaa gcatgcatct 4980 caattagtcagcaaccaggt gtggaaagtc cccaggctcc ccagcaggca gaagtatgca 5040 aagcatgcatctcaattagt cagcaaccat agtcccgccc ctaactccgc ccatcccgcc 5100 cctaactccgcccagttccg cccattctcc gccccatggc tgactaattt tttttattta 5160 tgcagaggccgaggccgcct ctgcctctga gctattccag aagtagtgag gaggcttttt 5220 tggaggcctaggcttttgca aaaagctccc gggagcttgt atatccattt tcggatctga 5280 tcagcacgtgttgacaatta atcatcggca tagtatatcg gcatagtata atacgacaag 5340 gtgaggaactaaaccatggc caagcctttg tctcaagaag aatccaccct cattgaaaga 5400 gcaacggctacaatcaacag catccccatc tctgaagact acagcgtcgc cagcgcagct 5460 ctctctagcgacggccgcat cttcactggt gtcaatgtat atcattttac tgggggacct 5520 tgtgcagaactcgtggtgct gggcactgct gctgctgcgg cagctggcaa cctgacttgt 5580 atcgtcgcgatcggaaatga gaacaggggc atcttgagcc cctgcggacg gtgccgacag 5640 gtgcttctcgatctgcatcc tgggatcaaa gccatagtga aggacagtga tggacagccg 5700 acggcagttgggattcgtga attgctgccc tctggttatg tgtgggaggg ctaagcacaa 5760 ttcgagctcggtacctttaa gaccaatgac ttacaaggca gctgtagatc ttagccactt 5820 tttaaaagaaaaggggggac tggaagggct aattcactcc caacgaagac aagatctgct 5880 ttttgcttgtactgggtctc tctggttaga ccagatctga gcctgggagc tctctggcta 5940 actagggaacccactgctta agcctcaata aagcttgcct tgagtgcttc aagtagtgtg 6000 tgcccgtctgttgtgtgact ctggtaacta gagatccctc agaccctttt agtcagtgtg 6060 gaaaatctctagcagtagta gttcatgtca tcttattatt cagtatttat aacttgcaaa 6120 gaaatgaatatcagagagtg agaggaactt gtttattgca gcttataatg gttacaaata 6180 aagcaatagcatcacaaatt tcacaaataa agcatttttt tcactgcatt ctagttgtgg 6240 tttgtccaaactcatcaatg tatcttatca tgtctggctc tagctatccc gcccctaact 6300 ccgcccatcccgcccctaac tccgcccagt tccgcccatt ctccgcccca tggctgacta 6360 attttttttatttatgcaga ggccgaggcc gcctcggcct ctgagctatt ccagaagtag 6420 tgaggaggcttttttggagg cctagggacg tacccaattc gccctatagt gagtcgtatt 6480 acgcgcgctcactggccgtc gttttacaac gtcgtgactg ggaaaaccct ggcgttaccc 6540 aacttaatcgccttgcagca catccccctt tcgccagctg gcgtaatagc gaagaggccc 6600 gcaccgatcgcccttcccaa cagttgcgca gcctgaatgg cgaatgggac gcgccctgta 6660 gcggcgcattaagcgcggcg ggtgtggtgg ttacgcgcag cgtgaccgct acacttgcca 6720 gcgccctagcgcccgctcct ttcgctttct tcccttcctt tctcgccacg ttcgccggct 6780 ttccccgtcaagctctaaat cgggggctcc ctttagggtt ccgatttagt gctttacggc 6840 acctcgaccccaaaaaactt gattagggtg atggttcacg tagtgggcca tcgccctgat 6900 agacggtttttcgccctttg acgttggagt ccacgttctt taatagtgga ctcttgttcc 6960 aaactggaacaacactcaac cctatctcgg tctattcttt tgatttataa gggattttgc 7020 cgatttcggcctattggtta aaaaatgagc tgatttaaca aaaatttaac gcgaatttta 7080 acaaaatattaacgcttaca atttaggtgg cacttttcgg ggaaatgtgc gcggaacccc 7140 tatttgtttatttttctaaa tacattcaaa tatgtatccg ctcatgagac aataaccctg 7200 ataaatgcttcaataatatt gaaaaaggaa gagtatgagt attcaacatt tccgtgtcgc 7260 ccttattcccttttttgcgg cattttgcct tcctgttttt gctcacccag aaacgctggt 7320 gaaagtaaaagatgctgaag atcagttggg tgcacgagtg ggttacatcg aactggatct 7380 caacagcggtaagatccttg agagttttcg ccccgaagaa cgttttccaa tgatgagcac 7440 ttttaaagttctgctatgtg gcgcggtatt atcccgtatt gacgccgggc aagagcaact 7500 cggtcgccgcatacactatt ctcagaatga cttggttgag tactcaccag tcacagaaaa 7560 gcatcttacggatggcatga cagtaagaga attatgcagt gctgccataa ccatgagtga 7620 taacactgcggccaacttac ttctgacaac gatcggagga ccgaaggagc taaccgcttt 7680 tttgcacaacatgggggatc atgtaactcg ccttgatcgt tgggaaccgg agctgaatga 7740 agccataccaaacgacgagc gtgacaccac gatgcctgta gcaatggcaa caacgttgcg 7800 caaactattaactggcgaac tacttactct agcttcccgg caacaattaa tagactggat 7860 ggaggcggataaagttgcag gaccacttct gcgctcggcc cttccggctg gctggtttat 7920 tgctgataaatctggagccg gtgagcgtgg gtctcgcggt atcattgcag cactggggcc 7980 agatggtaagccctcccgta tcgtagttat ctacacgacg gggagtcagg caactatgga 8040 tgaacgaaatagacagatcg ctgagatagg tgcctcactg attaagcatt ggtaactgtc 8100 agaccaagtttactcatata tactttagat tgatttaaaa cttcattttt aatttaaaag 8160 gatctaggtgaagatccttt ttgataatct catgaccaaa atcccttaac gtgagttttc 8220 gttccactgagcgtcagacc ccgtagaaaa gatcaaagga tcttcttgag atcctttttt 8280 tctgcgcgtaatctgctgct tgcaaacaaa aaaaccaccg ctaccagcgg tggtttgttt 8340 gccggatcaagagctaccaa ctctttttcc gaaggtaact ggcttcagca gagcgcagat 8400 accaaatactgttcttctag tgtagccgta gttaggccac cacttcaaga actctgtagc 8460 accgcctacatacctcgctc tgctaatcct gttaccagtg gctgctgcca gtggcgataa 8520 gtcgtgtcttaccgggttgg actcaagacg atagttaccg gataaggcgc agcggtcggg 8580 ctgaacggggggttcgtgca cacagcccag cttggagcga acgacctaca ccgaactgag 8640 atacctacagcgtgagctat gagaaagcgc cacgcttccc gaagggagaa aggcggacag 8700 gtatccggtaagcggcaggg tcggaacagg agagcgcacg agggagcttc cagggggaaa 8760 cgcctggtatctttatagtc ctgtcgggtt tcgccacctc tgacttgagc gtcgattttt 8820 gtgatgctcgtcaggggggc ggagcctatg gaaaaacgcc agcaacgcgg cctttttacg 8880 gttcctggccttttgctggc cttttgctca catgttcttt cctgcgttat cccctgattc 8940 tgtggataaccgtattaccg cctttgagtg agctgatacc gctcgccgca gccgaacgac 9000 cgagcgcagcgagtcagtga gcgaggaagc ggaagagcgc ccaatacgca aaccgcctct 9060 ccccgcgcgttggccgattc attaatgcag ctggcacgac aggtttcccg actggaaagc 9120 gggcagtgagcgcaacgcaa ttaatgtgag ttagctcact cattaggcac cccaggcttt 9180 acactttatgcttccggctc gtatgttgtg tggaattgtg agcggataac aatttcacac 9240 aggaaacagctatgaccatg attacgccaa gcgcgcaatt aaccctcact aaagggaaca 9300 aaagctggagctgcaagctt 9320 109 8889 DNA Artificial Sequence pLP1 109 ttggcccattgcatacgttg tatccatatc ataatatgta catttatatt ggctcatgtc 60 caacattaccgccatgttga cattgattat tgactagtta ttaatagtaa tcaattacgg 120 ggtcattagttcatagccca tatatggagt tccgcgttac ataacttacg gtaaatggcc 180 cgcctggctgaccgcccaac gacccccgcc cattgacgtc aataatgacg tatgttccca 240 tagtaacgccaatagggact ttccattgac gtcaatgggt ggagtattta cggtaaactg 300 cccacttggcagtacatcaa gtgtatcata tgccaagtac gccccctatt gacgtcaatg 360 acggtaaatggcccgcctgg cattatgccc agtacatgac cttatgggac tttcctactt 420 ggcagtacatctacgtatta gtcatcgcta ttaccatggt gatgcggttt tggcagtaca 480 tcaatgggcgtggatagcgg tttgactcac ggggatttcc aagtctccac cccattgacg 540 tcaatgggagtttgttttgg caccaaaatc aacgggactt tccaaaatgt cgtaacaact 600 ccgccccattgacgcaaatg ggcggtaggc gtgtacggtg ggaggtctat ataagcagag 660 ctcgtttagtgaaccgtcag atcgcctgga gacgccatcc acgctgtttt gacctccata 720 gaagacaccgggaccgatcc agcctcccct cgaagcttac atgtggtacc gagctcggat 780 cctgagaacttcagggtgag tctatgggac ccttgatgtt ttctttcccc ttcttttcta 840 tggttaagttcatgtcatag gaaggggaga agtaacaggg tacacatatt gaccaaatca 900 gggtaattttgcatttgtaa ttttaaaaaa tgctttcttc ttttaatata cttttttgtt 960 tatcttatttctaatacttt ccctaatctc tttctttcag ggcaataatg atacaatgta 1020 tcatgcctctttgcaccatt ctaaagaata acagtgataa tttctgggtt aaggcaatag 1080 caatatttctgcatataaat atttctgcat ataaattgta actgatgtaa gaggtttcat 1140 attgctaatagcagctacaa tccagctacc attctgcttt tattttatgg ttgggataag 1200 gctggattattctgagtcca agctaggccc ttttgctaat catgttcata cctcttatct 1260 tcctcccacagctcctgggc aacgtgctgg tctgtgtgct ggcccatcac tttggcaaag 1320 cacgtgagatctgaattcga gatctgccgc cgccatgggt gcgagagcgt cagtattaag 1380 cgggggagaattagatcgat gggaaaaaat tcggttaagg ccagggggaa agaaaaaata 1440 taaattaaaacatatagtat gggcaagcag ggagctagaa cgattcgcag ttaatcctgg 1500 cctgttagaaacatcagaag gctgtagaca aatactggga cagctacaac catcccttca 1560 gacaggatcagaagaactta gatcattata taatacagta gcaaccctct attgtgtgca 1620 tcaaaggatagagataaaag acaccaagga agctttagac aagatagagg aagagcaaaa 1680 caaaagtaagaaaaaagcac agcaagcagc agctgacaca ggacacagca atcaggtcag 1740 ccaaaattaccctatagtgc agaacatcca ggggcaaatg gtacatcagg ccatatcacc 1800 tagaactttaaatgcatggg taaaagtagt agaagagaag gctttcagcc cagaagtgat 1860 acccatgttttcagcattat cagaaggagc caccccacaa gatttaaaca ccatgctaaa 1920 cacagtggggggacatcaag cagccatgca aatgttaaaa gagaccatca atgaggaagc 1980 tgcagaatgggatagagtgc atccagtgca tgcagggcct attgcaccag gccagatgag 2040 agaaccaaggggaagtgaca tagcaggaac tactagtacc cttcaggaac aaataggatg 2100 gatgacacataatccaccta tcccagtagg agaaatctat aaaagatgga taatcctggg 2160 attaaataaaatagtaagaa tgtatagccc taccagcatt ctggacataa gacaaggacc 2220 aaaggaaccctttagagact atgtagaccg attctataaa actctaagag ccgagcaagc 2280 ttcacaagaggtaaaaaatt ggatgacaga aaccttgttg gtccaaaatg cgaacccaga 2340 ttgtaagactattttaaaag cattgggacc aggagcgaca ctagaagaaa tgatgacagc 2400 atgtcagggagtggggggac ccggccataa agcaagagtt ttggctgaag caatgagcca 2460 agtaacaaatccagctacca taatgataca gaaaggcaat tttaggaacc aaagaaagac 2520 tgttaagtgtttcaattgtg gcaaagaagg gcacatagcc aaaaattgca gggcccctag 2580 gaaaaagggctgttggaaat gtggaaagga aggacaccaa atgaaagatt gtactgagag 2640 acaggctaattttttaggga agatctggcc ttcccacaag ggaaggccag ggaattttct 2700 tcagagcagaccagagccaa cagccccacc agaagagagc ttcaggtttg gggaagagac 2760 aacaactccctctcagaagc aggagccgat agacaaggaa ctgtatcctt tagcttccct 2820 cagatcactctttggcagcg acccctcgtc acaataaaga taggggggca attaaaggaa 2880 gctctattagatacaggagc agatgataca gtattagaag aaatgaattt gccaggaaga 2940 tggaaaccaaaaatgatagg gggaattgga ggttttatca aagtaagaca gtatgatcag 3000 atactcatagaaatctgcgg acataaagct ataggtacag tattagtagg acctacacct 3060 gtcaacataattggaagaaa tctgttgact cagattggct gcactttaaa ttttcccatt 3120 agtcctattgagactgtacc agtaaaatta aagccaggaa tggatggccc aaaagttaaa 3180 caatggccattgacagaaga aaaaataaaa gcattagtag aaatttgtac agaaatggaa 3240 aaggaaggaaaaatttcaaa aattgggcct gaaaatccat acaatactcc agtatttgcc 3300 ataaagaaaaaagacagtac taaatggaga aaattagtag atttcagaga acttaataag 3360 agaactcaagatttctggga agttcaatta ggaataccac atcctgcagg gttaaaacag 3420 aaaaaatcagtaacagtact ggatgtgggc gatgcatatt tttcagttcc cttagataaa 3480 gacttcaggaagtatactgc atttaccata cctagtataa acaatgagac accagggatt 3540 agatatcagtacaatgtgct tccacaggga tggaaaggat caccagcaat attccagtgt 3600 agcatgacaaaaatcttaga gccttttaga aaacaaaatc cagacatagt catctatcaa 3660 tacatggatgatttgtatgt aggatctgac ttagaaatag ggcagcatag aacaaaaata 3720 gaggaactgagacaacatct gttgaggtgg ggatttacca caccagacaa aaaacatcag 3780 aaagaacctccattcctttg gatgggttat gaactccatc ctgataaatg gacagtacag 3840 cctatagtgctgccagaaaa ggacagctgg actgtcaatg acatacagaa attagtggga 3900 aaattgaattgggcaagtca gatttatgca gggattaaag taaggcaatt atgtaaactt 3960 cttaggggaaccaaagcact aacagaagta gtaccactaa cagaagaagc agagctagaa 4020 ctggcagaaaacagggagat tctaaaagaa ccggtacatg gagtgtatta tgacccatca 4080 aaagacttaatagcagaaat acagaagcag gggcaaggcc aatggacata tcaaatttat 4140 caagagccatttaaaaatct gaaaacagga aagtatgcaa gaatgaaggg tgcccacact 4200 aatgatgtgaaacaattaac agaggcagta caaaaaatag ccacagaaag catagtaata 4260 tggggaaagactcctaaatt taaattaccc atacaaaagg aaacatggga agcatggtgg 4320 acagagtattggcaagccac ctggattcct gagtgggagt ttgtcaatac ccctccctta 4380 gtgaagttatggtaccagtt agagaaagaa cccataatag gagcagaaac tttctatgta 4440 gatggggcagccaataggga aactaaatta ggaaaagcag gatatgtaac tgacagagga 4500 agacaaaaagttgtccccct aacggacaca acaaatcaga agactgagtt acaagcaatt 4560 catctagctttgcaggattc gggattagaa gtaaacatag tgacagactc acaatatgca 4620 ttgggaatcattcaagcaca accagataag agtgaatcag agttagtcag tcaaataata 4680 gagcagttaataaaaaagga aaaagtctac ctggcatggg taccagcaca caaaggaatt 4740 ggaggaaatgaacaagtaga taaattggtc agtgctggaa tcaggaaagt actattttta 4800 gatggaatagataaggccca agaagaacat gagaaatatc acagtaattg gagagcaatg 4860 gctagtgattttaacctacc acctgtagta gcaaaagaaa tagtagccag ctgtgataaa 4920 tgtcagctaaaaggggaagc catgcatgga caagtagact gtagcccagg aatatggcag 4980 ctagattgtacacatttaga aggaaaagtt atcttggtag cagttcatgt agccagtgga 5040 tatatagaagcagaagtaat tccagcagag acagggcaag aaacagcata cttcctctta 5100 aaattagcaggaagatggcc agtaaaaaca gtacatacag acaatggcag caatttcacc 5160 agtactacagttaaggccgc ctgttggtgg gcggggatca agcaggaatt tggcattccc 5220 tacaatccccaaagtcaagg agtaatagaa tctatgaata aagaattaaa gaaaattata 5280 ggacaggtaagagatcaggc tgaacatctt aagacagcag tacaaatggc agtattcatc 5340 cacaattttaaaagaaaagg ggggattggg gggtacagtg caggggaaag aatagtagac 5400 ataatagcaacagacataca aactaaagaa ttacaaaaac aaattacaaa aattcaaaat 5460 tttcgggtttattacaggga cagcagagat ccagtttgga aaggaccagc aaagctcctc 5520 tggaaaggtgaaggggcagt agtaatacaa gataatagtg acataaaagt agtgccaaga 5580 agaaaagcaaagatcatcag ggattatgga aaacagatgg caggtgatga ttgtgtggca 5640 agtagacaggatgaggatta acacatggaa ttccggagcg gccgcaggag ctttgttcct 5700 tgggttcttgggagcagcag gaagcactat gggcgcagcg tcaatgacgc tgacggtaca 5760 ggccagacaattattgtctg gtatagtgca gcagcagaac aatttgctga gggctattga 5820 ggcgcaacagcatctgttgc aactcacagt ctggggcatc aagcagctcc aggcaagaat 5880 cctggctgtggaaagatacc taaaggatca acagctcctg gggatttggg gttgctctgg 5940 aaaactcatttgcaccactg ctgtgccttg gaatgctagt tggagtaata aatctctgga 6000 acagatttggaatcacacga cctggatgga gtgggacaga gaaattaaca attacacaag 6060 cttccgcggaattcacccca ccagtgcagg ctgcctatca gaaagtggtg gctggtgtgg 6120 ctaatgccctggcccacaag tatcactaag ctcgctttct tgctgtccaa tttctattaa 6180 aggttcctttgttccctaag tccaactact aaactggggg atattatgaa gggccttgag 6240 catctggattctgcctaata aaaaacattt attttcattg caatgatgta tttaaattat 6300 ttctgaatattttactaaaa agggaatgtg ggaggtcagt gcatttaaaa cataaagaaa 6360 tgaagagctagttcaaacct tgggaaaata cactatatct taaactccat gaaagaaggt 6420 gaggctgcaaacagctaatg cacattggca acagcccctg atgcctatgc cttattcatc 6480 cctcagaaaaggattcaagt agaggcttga tttggaggtt aaagttttgc tatgctgtat 6540 tttacattacttattgtttt agctgtcctc atgaatgtct tttcactacc catttgctta 6600 tcctgcatctctcagccttg actccactca gttctcttgc ttagagatac cacctttccc 6660 ctgaagtgttccttccatgt tttacggcga gatggtttct cctcgcctgg ccactcagcc 6720 ttagttgtctctgttgtctt atagaggtct acttgaagaa ggaaaaacag ggggcatggt 6780 ttgactgtcctgtgagccct tcttccctgc ctcccccact cacagtgacc cggaatccct 6840 cgacatggcagtctagcact agtgcggccg cagatctgct tcctcgctca ctgactcgct 6900 gcgctcggtcgttcggctgc ggcgagcggt atcagctcac tcaaaggcgg taatacggtt 6960 atccacagaatcaggggata acgcaggaaa gaacatgtga gcaaaaggcc agcaaaaggc 7020 caggaaccgtaaaaaggccg cgttgctggc gtttttccat aggctccgcc cccctgacga 7080 gcatcacaaaaatcgacgct caagtcagag gtggcgaaac ccgacaggac tataaagata 7140 ccaggcgtttccccctggaa gctccctcgt gcgctctcct gttccgaccc tgccgcttac 7200 cggatacctgtccgcctttc tcccttcggg aagcgtggcg ctttctcata gctcacgctg 7260 taggtatctcagttcggtgt aggtcgttcg ctccaagctg ggctgtgtgc acgaaccccc 7320 cgttcagcccgaccgctgcg ccttatccgg taactatcgt cttgagtcca acccggtaag 7380 acacgacttatcgccactgg cagcagccac tggtaacagg attagcagag cgaggtatgt 7440 aggcggtgctacagagttct tgaagtggtg gcctaactac ggctacacta gaagaacagt 7500 atttggtatctgcgctctgc tgaagccagt taccttcgga aaaagagttg gtagctcttg 7560 atccggcaaacaaaccaccg ctggtagcgg tggttttttt gtttgcaagc agcagattac 7620 gcgcagaaaaaaaggatctc aagaagatcc tttgatcttt tctacggggt ctgacgctca 7680 gtggaacgaaaactcacgtt aagggatttt ggtcatgaga ttatcaaaaa ggatcttcac 7740 ctagatccttttaaattaaa aatgaagttt taaatcaatc taaagtatat atgagtaaac 7800 ttggtctgacagttaccaat gcttaatcag tgaggcacct atctcagcga tctgtctatt 7860 tcgttcatccatagttgcct gactccccgt cgtgtagata actacgatac gggagggctt 7920 accatctggccccagtgctg caatgatacc gcgagaccca cgctcaccgg ctccagattt 7980 atcagcaataaaccagccag ccggaagggc cgagcgcaga agtggtcctg caactttatc 8040 cgcctccatccagtctatta attgttgccg ggaagctaga gtaagtagtt cgccagttaa 8100 tagtttgcgcaacgttgttg ccattgctac aggcatcgtg gtgtcacgct cgtcgtttgg 8160 tatggcttcattcagctccg gttcccaacg atcaaggcga gttacatgat cccccatgtt 8220 gtgcaaaaaagcggttagct ccttcggtcc tccgatcgtt gtcagaagta agttggccgc 8280 agtgttatcactcatggtta tggcagcact gcataattct cttactgtca tgccatccgt 8340 aagatgcttttctgtgactg gtgagtactc aaccaagtca ttctgagaat agtgtatgcg 8400 gcgaccgagttgctcttgcc cggcgtcaat acgggataat accgcgccac atagcagaac 8460 tttaaaagtgctcatcattg gaaaacgttc ttcggggcga aaactctcaa ggatcttacc 8520 gctgttgagatccagttcga tgtaacccac tcgtgcaccc aactgatctt cagcatcttt 8580 tactttcaccagcgtttctg ggtgagcaaa aacaggaagg caaaatgccg caaaaaaggg 8640 aataagggcgacacggaaat gttgaatact catactcttc ctttttcaat attattgaag 8700 catttatcagggttattgtc tcatgagcgg atacatattt gaatgtattt agaaaaataa 8760 acaaataggggttccgcgca catttccccg aaaagtgcca cctgacggga tcccctgagg 8820 gggcccccatgggctagagg atccggcctc ggcctctgca taaataaaaa aaattagtca 8880 gccatgagc8889 110 4180 DNA Artificial Sequence pLP2 110 aatgtagtct tatgcaatactcttgtagtc ttgcaacatg gtaacgatga gttagcaaca 60 tgccttacaa ggagagaaaaagcaccgtgc atgccgattg gtggaagtaa ggtggtacga 120 tcgtgcctta ttaggaaggcaacagacggg tctgacatgg attggacgaa ccactgaatt 180 ccgcattgca gagatattgtatttaagtgc ctagctcgat acaataaacg ccatttgacc 240 attcaccaca ttggtgtgcacctccaagct cgagctcgtt tagtgaaccg tcagatcgcc 300 tggagacgcc atccacgctgttttgacctc catagaagac accgggaccg atccagcctc 360 ccctcgaagc tagtcgattaggcatctcct atggcaggaa gaagcggaga cagcgacgaa 420 gacctcctca aggcagtcagactcatcaag tttctctatc aaagcaaccc acctcccaat 480 cccgagggga cccgacaggcccgaaggaat agaagaagaa ggtggagaga gagacagaga 540 cagatccatt cgattagtgaacggatcctt agcacttatc tgggacgatc tgcggagcct 600 gtgcctcttc agctaccaccgcttgagaga cttactcttg attgtaacga ggattgtgga 660 acttctggga cgcagggggtgggaagccct caaatattgg tggaatctcc tacaatattg 720 gagtcaggag ctaaagaatagtgctgttag cttgctcaat gccacagcta tagcagtagc 780 tgaggggaca gatagggttatagaagtagt acaagaagct tggcactggc cgtcgtttta 840 caacgtcgtg atctgagcctgggagatctc tggctaacta gggaacccac tgcttaagcc 900 tcaataaagc ttgccttgagtgcttcaagt agtgtgtgcc cgtctgttgt gtgactctgg 960 taactagaga tcaggaaaaccctggcgtta cccaacttaa tcgccttgca gcacatcccc 1020 ctttcgccag ctggcgtaatagcgaagagg cccgcaccga tcgcccttcc caacagttgc 1080 gcagcctgaa tggcgaatggcgcctgatgc ggtattttct ccttacgcat ctgtgcggta 1140 tttcacaccg catacgtcaaagcaaccata gtacgcgccc tgtagcggcg cattaagcgc 1200 ggcgggtgtg gtggttacgcgcagcgtgac cgctacactt gccagcgccc tagcgcccgc 1260 tcctttcgct ttcttcccttcctttctcgc cacgttcgcc ggctttcccc gtcaagctct 1320 aaatcggggg ctccctttagggttccgatt tagtgcttta cggcacctcg accccaaaaa 1380 acttgatttg ggtgatggttcacgtagtgg gccatcgccc tgatagacgg tttttcgccc 1440 tttgacgttg gagtccacgttctttaatag tggactcttg ttccaaactg gaacaacact 1500 caaccctatc tcgggctattcttttgattt ataagggatt ttgccgattt cggcctattg 1560 gttaaaaaat gagctgatttaacaaaaatt taacgcgaat tttaacaaaa tattaacgtt 1620 tacaatttta tggtgcactctcagtacaat ctgctctgat gccgcatagt taagccagcc 1680 ccgacacccg ccaacacccgctgacgcgcc ctgacgggct tgtctgctcc cggcatccgc 1740 ttacagacaa gctgtgaccgtctccgggag ctgcatgtgt cagaggtttt caccgtcatc 1800 accgaaacgc gcgagacgaaagggcctcgt gatacgccta tttttatagg ttaatgtcat 1860 gataataatg gtttcttagacgtcaggtgg cacttttcgg ggaaatgtgc gcggaacccc 1920 tatttgttta tttttctaaatacattcaaa tatgtatccg ctcatgagac aataaccctg 1980 ataaatgctt caataatattgaaaaaggaa gagtatgagt attcaacatt tccgtgtcgc 2040 ccttattccc ttttttgcggcattttgcct tcctgttttt gctcacccag aaacgctggt 2100 gaaagtaaaa gatgctgaagatcagttggg tgcacgagtg ggttacatcg aactggatct 2160 caacagcggt aagatccttgagagttttcg ccccgaagaa cgttttccaa tgatgagcac 2220 ttttaaagtt ctgctatgtggcgcggtatt atcccgtatt gacgccgggc aagagcaact 2280 cggtcgccgc atacactattctcagaatga cttggttgag tactcaccag tcacagaaaa 2340 gcatcttacg gatggcatgacagtaagaga attatgcagt gctgccataa ccatgagtga 2400 taacactgcg gccaacttacttctgacaac gatcggagga ccgaaggagc taaccgcttt 2460 tttgcacaac atgggggatcatgtaactcg ccttgatcgt tgggaaccgg agctgaatga 2520 agccatacca aacgacgagcgtgacaccac gatgcctgta gcaatggcaa caacgttgcg 2580 caaactatta actggcgaactacttactct agcttcccgg caacaattaa tagactggat 2640 ggaggcggat aaagttgcaggaccacttct gcgctcggcc cttccggctg gctggtttat 2700 tgctgataaa tctggagccggtgagcgtgg gtctcgcggt atcattgcag cactggggcc 2760 agatggtaag ccctcccgtatcgtagttat ctacacgacg gggagtcagg caactatgga 2820 tgaacgaaat agacagatcgctgagatagg tgcctcactg attaagcatt ggtaactgtc 2880 agaccaagtt tactcatatatactttagat tgatttaaaa cttcattttt aatttaaaag 2940 gatctaggtg aagatcctttttgataatct catgaccaaa atcccttaac gtgagttttc 3000 gttccactga gcgtcagaccccgtagaaaa gatcaaagga tcttcttgag atcctttttt 3060 tctgcgcgta atctgctgcttgcaaacaaa aaaaccaccg ctaccagcgg tggtttgttt 3120 gccggatcaa gagctaccaactctttttcc gaaggtaact ggcttcagca gagcgcagat 3180 accaaatact gttcttctagtgtagccgta gttaggccac cacttcaaga actctgtagc 3240 accgcctaca tacctcgctctgctaatcct gttaccagtg gctgctgcca gtggcgataa 3300 gtcgtgtctt accgggttggactcaagacg atagttaccg gataaggcgc agcggtcggg 3360 ctgaacgggg ggttcgtgcacacagcccag cttggagcga acgacctaca ccgaactgag 3420 atacctacag cgtgagctatgagaaagcgc cacgcttccc gaagggagaa aggcggacag 3480 gtatccggta agcggcagggtcggaacagg agagcgcacg agggagcttc cagggggaaa 3540 cgcctggtat ctttatagtcctgtcgggtt tcgccacctc tgacttgagc gtcgattttt 3600 gtgatgctcg tcaggggggcggagcctatg gaaaaacgcc agcaacgcgg cctttttacg 3660 gttcctggcc ttttgctggccttttgctca catgttcttt cctgcgttat cccctgattc 3720 tgtggataac cgtattaccgcctttgagtg agctgatacc gctcgccgca gccgaacgac 3780 cgagcgcagc gagtcagtgagcgaggaagc ggaagagcgc ccaatacgca aaccgcctct 3840 ccccgcgcgt tggccgattcattaatgcag ctggcacgac aggtttcccg actggaaagc 3900 gggcagtgag cgcaacgcaattaatgtgag ttagctcact cattaggcac cccaggcttt 3960 acactttatg cttccggctcgtatgttgtg tggaattgtg agcggataac aatttcacac 4020 aggaaacagc tatgacatgattacgaattc gatgtacggg ccagatatac gcgtatctga 4080 ggggactagg gtgtgtttaggcgaaaagcg gggcttcggt tgtacgcggt taggagtccc 4140 ctcaggatat agtagtttcgcttttgcata gggaggggga 4180 111 5821 DNA Artificial Sequence pLP/VSVG 111ttggcccatt gcatacgttg tatccatatc ataatatgta catttatatt ggctcatgtc 60caacattacc gccatgttga cattgattat tgactagtta ttaatagtaa tcaattacgg 120ggtcattagt tcatagccca tatatggagt tccgcgttac ataacttacg gtaaatggcc 180cgcctggctg accgcccaac gacccccgcc cattgacgtc aataatgacg tatgttccca 240tagtaacgcc aatagggact ttccattgac gtcaatgggt ggagtattta cggtaaactg 300cccacttggc agtacatcaa gtgtatcata tgccaagtac gccccctatt gacgtcaatg 360acggtaaatg gcccgcctgg cattatgccc agtacatgac cttatgggac tttcctactt 420ggcagtacat ctacgtatta gtcatcgcta ttaccatggt gatgcggttt tggcagtaca 480tcaatgggcg tggatagcgg tttgactcac ggggatttcc aagtctccac cccattgacg 540tcaatgggag tttgttttgg caccaaaatc aacgggactt tccaaaatgt cgtaacaact 600ccgccccatt gacgcaaatg ggcggtaggc gtgtacggtg ggaggtctat ataagcagag 660ctcgtttagt gaaccgtcag atcgcctgga gacgccatcc acgctgtttt gacctccata 720gaagacaccg ggaccgatcc agcctcccct cgaagcttac atgtggtacc gagctcggat 780cctgagaact tcagggtgag tctatgggac ccttgatgtt ttctttcccc ttcttttcta 840tggttaagtt catgtcatag gaaggggaga agtaacaggg tacacatatt gaccaaatca 900gggtaatttt gcatttgtaa ttttaaaaaa tgctttcttc ttttaatata cttttttgtt 960tatcttattt ctaatacttt ccctaatctc tttctttcag ggcaataatg atacaatgta 1020tcatgcctct ttgcaccatt ctaaagaata acagtgataa tttctgggtt aaggcaatag 1080caatatttct gcatataaat atttctgcat ataaattgta actgatgtaa gaggtttcat 1140attgctaata gcagctacaa tccagctacc attctgcttt tattttatgg ttgggataag 1200gctggattat tctgagtcca agctaggccc ttttgctaat catgttcata cctcttatct 1260tcctcccaca gctcctgggc aacgtgctgg tctgtgtgct ggcccatcac tttggcaaag 1320cacgtgagat ctgaattctg acactatgaa gtgccttttg tacttagcct ttttattcat 1380tggggtgaat tgcaagttca ccatagtttt tccacacaac caaaaaggaa actggaaaaa 1440tgttccttct aattaccatt attgcccgtc aagctcagat ttaaattggc ataatgactt 1500aataggcaca gccttacaag tcaaaatgcc caagagtcac aaggctattc aagcagacgg 1560ttggatgtgt catgcttcca aatgggtcac tacttgtgat ttccgctggt atggaccgaa 1620gtatataaca cattccatcc gatccttcac tccatctgta gaacaatgca aggaaagcat 1680tgaacaaacg aaacaaggaa cttggctgaa tccaggcttc cctcctcaaa gttgtggata 1740tgcaactgtg acggatgccg aagcagtgat tgtccaggtg actcctcacc atgtgctggt 1800tgatgaatac acaggagaat gggttgattc acagttcatc aacggaaaat gcagcaatta 1860catatgcccc actgtccata actctacaac ctggcattct gactataagg tcaaagggct 1920atgtgattct aacctcattt ccatggacat caccttcttc tcagaggacg gagagctatc 1980atccctggga aaggagggca cagggttcag aagtaactac tttgcttatg aaactggagg 2040caaggcctgc aaaatgcaat actgcaagca ttggggagtc agactcccat caggtgtctg 2100gttcgagatg gctgataagg atctctttgc tgcagccaga ttccctgaat gcccagaagg 2160gtcaagtatc tctgctccat ctcagacctc agtggatgta agtctaattc aggacgttga 2220gaggatcttg gattattccc tctgccaaga aacctggagc aaaatcagag cgggtcttcc 2280aatctctcca gtggatctca gctatcttgc tcctaaaaac ccaggaaccg gtcctgcttt 2340caccataatc aatggtaccc taaaatactt tgagaccaga tacatcagag tcgatattgc 2400tgctccaatc ctctcaagaa tggtcggaat gatcagtgga actaccacag aaagggaact 2460gtgggatgac tgggcaccat atgaagacgt ggaaattgga cccaatggag ttctgaggac 2520cagttcagga tataagtttc ctttatacat gattggacat ggtatgttgg actccgatct 2580tcatcttagc tcaaaggctc aggtgttcga acatcctcac attcaagacg ctgcttcgca 2640acttcctgat gatgagagtt tattttttgg tgatactggg ctatccaaaa atccaatcga 2700gcttgtagaa ggttggttca gtagttggaa aagctctatt gcctcttttt tctttatcat 2760agggttaatc attggactat tcttggttct ccgagttggt atccatcttt gcattaaatt 2820aaagcacacc aagaaaagac agatttatac agacatagag atgaaccgac ttggaaagta 2880actcaaatcc tgcacaacag attcttcatg tttggaccaa atcaacttgt gataccatgc 2940tcaaagaggc ctcaattata tttgagtttt taatttttat gaaaaaaaaa aaaaaaaacg 3000gaattcaccc caccagtgca ggctgcctat cagaaagtgg tggctggtgt ggctaatgcc 3060ctggcccaca agtatcacta agctcgcttt cttgctgtcc aatttctatt aaaggttcct 3120ttgttcccta agtccaacta ctaaactggg ggatattatg aagggccttg agcatctgga 3180ttctgcctaa taaaaaacat ttattttcat tgcaatgatg tatttaaatt atttctgaat 3240attttactaa aaagggaatg tgggaggtca gtgcatttaa aacataaaga aatgaagagc 3300tagttcaaac cttgggaaaa tacactatat cttaaactcc atgaaagaag gtgaggctgc 3360aaacagctaa tgcacattgg caacagcccc tgatgcctat gccttattca tccctcagaa 3420aaggattcaa gtagaggctt gatttggagg ttaaagtttt gctatgctgt attttacatt 3480acttattgtt ttagctgtcc tcatgaatgt cttttcacta cccatttgct tatcctgcat 3540ctctcagcct tgactccact cagttctctt gcttagagat accacctttc ccctgaagtg 3600ttccttccat gttttacggc gagatggttt ctcctcgcct ggccactcag ccttagttgt 3660ctctgttgtc ttatagaggt ctacttgaag aaggaaaaac agggggcatg gtttgactgt 3720cctgtgagcc cttcttccct gcctccccca ctcacagtga cccggaatcc ctcgacatgg 3780cagtctagca ctagtgcggc cgcagatctg cttcctcgct cactgactcg ctgcgctcgg 3840tcgttcggct gcggcgagcg gtatcagctc actcaaaggc ggtaatacgg ttatccacag 3900aatcagggga taacgcagga aagaacatgt gagcaaaagg ccagcaaaag gccaggaacc 3960gtaaaaaggc cgcgttgctg gcgtttttcc ataggctccg cccccctgac gagcatcaca 4020aaaatcgacg ctcaagtcag aggtggcgaa acccgacagg actataaaga taccaggcgt 4080ttccccctgg aagctccctc gtgcgctctc ctgttccgac cctgccgctt accggatacc 4140tgtccgcctt tctcccttcg ggaagcgtgg cgctttctca tagctcacgc tgtaggtatc 4200tcagttcggt gtaggtcgtt cgctccaagc tgggctgtgt gcacgaaccc cccgttcagc 4260ccgaccgctg cgccttatcc ggtaactatc gtcttgagtc caacccggta agacacgact 4320tatcgccact ggcagcagcc actggtaaca ggattagcag agcgaggtat gtaggcggtg 4380ctacagagtt cttgaagtgg tggcctaact acggctacac tagaagaaca gtatttggta 4440tctgcgctct gctgaagcca gttaccttcg gaaaaagagt tggtagctct tgatccggca 4500aacaaaccac cgctggtagc ggtggttttt ttgtttgcaa gcagcagatt acgcgcagaa 4560aaaaaggatc tcaagaagat cctttgatct tttctacggg gtctgacgct cagtggaacg 4620aaaactcacg ttaagggatt ttggtcatga gattatcaaa aaggatcttc acctagatcc 4680ttttaaatta aaaatgaagt tttaaatcaa tctaaagtat atatgagtaa acttggtctg 4740acagttacca atgcttaatc agtgaggcac ctatctcagc gatctgtcta tttcgttcat 4800ccatagttgc ctgactcccc gtcgtgtaga taactacgat acgggagggc ttaccatctg 4860gccccagtgc tgcaatgata ccgcgagacc cacgctcacc ggctccagat ttatcagcaa 4920taaaccagcc agccggaagg gccgagcgca gaagtggtcc tgcaacttta tccgcctcca 4980tccagtctat taattgttgc cgggaagcta gagtaagtag ttcgccagtt aatagtttgc 5040gcaacgttgt tgccattgct acaggcatcg tggtgtcacg ctcgtcgttt ggtatggctt 5100cattcagctc cggttcccaa cgatcaaggc gagttacatg atcccccatg ttgtgcaaaa 5160aagcggttag ctccttcggt cctccgatcg ttgtcagaag taagttggcc gcagtgttat 5220cactcatggt tatggcagca ctgcataatt ctcttactgt catgccatcc gtaagatgct 5280tttctgtgac tggtgagtac tcaaccaagt cattctgaga atagtgtatg cggcgaccga 5340gttgctcttg cccggcgtca atacgggata ataccgcgcc acatagcaga actttaaaag 5400tgctcatcat tggaaaacgt tcttcggggc gaaaactctc aaggatctta ccgctgttga 5460gatccagttc gatgtaaccc actcgtgcac ccaactgatc ttcagcatct tttactttca 5520ccagcgtttc tgggtgagca aaaacaggaa ggcaaaatgc cgcaaaaaag ggaataaggg 5580cgacacggaa atgttgaata ctcatactct tcctttttca atattattga agcatttatc 5640agggttattg tctcatgagc ggatacatat ttgaatgtat ttagaaaaat aaacaaatag 5700gggttccgcg cacatttccc cgaaaagtgc cacctgacgg gatcccctga gggggccccc 5760atgggctaga ggatccggcc tcggcctctg cataaataaa aaaaattagt cagccatgag 5820 c5821 112 7341 DNA Artificial Sequence pcDNA6.2/V5-DEST 112 gacggatcgggagatctccc gatcccctat ggtgcactct cagtacaatc tgctctgatg 60 ccgcatagttaagccagtat ctgctccctg cttgtgtgtt ggaggtcgct gagtagtgcg 120 cgagcaaaatttaagctaca acaaggcaag gcttgaccga caattgcatg aagaatctgc 180 ttagggttaggcgttttgcg ctgcttcgcg atgtacgggc cagatatacg cgttgacatt 240 gattattgactagttattaa tagtaatcaa ttacggggtc attagttcat agcccatata 300 tggagttccgcgttacataa cttacggtaa atggcccgcc tggctgaccg cccaacgacc 360 cccgcccattgacgtcaata atgacgtatg ttcccatagt aacgccaata gggactttcc 420 attgacgtcaatgggtggag tatttacggt aaactgccca cttggcagta catcaagtgt 480 atcatatgccaagtacgccc cctattgacg tcaatgacgg taaatggccc gcctggcatt 540 atgcccagtacatgacctta tgggactttc ctacttggca gtacatctac gtattagtca 600 tcgctattaccatggtgatg cggttttggc agtacatcaa tgggcgtgga tagcggtttg 660 actcacggggatttccaagt ctccacccca ttgacgtcaa tgggagtttg ttttggcacc 720 aaaatcaacgggactttcca aaatgtcgta acaactccgc cccattgacg caaatgggcg 780 gtaggcgtgtacggtgggag gtctatataa gcagagctct ctggctaact agagaaccca 840 ctgcttactggcttatcgaa attaatacga ctcactatag ggagacccaa gctggctagt 900 taagctatcaacaagtttgt acaaaaaagc tgaacgagaa acgtaaaatg atataaatat 960 caatatattaaattagattt tgcataaaaa acagactaca taatactgta aaacacaaca 1020 tatccagtcactatgaatca actacttaga tggtattagt gacctgtagt cgaccgacag 1080 ccttccaaatgttcttcggg tgatgctgcc aacttagtcg accgacagcc ttccaaatgt 1140 tcttctcaaacggaatcgtc gtatccagcc tactcgctat tgtcctcaat gccgtattaa 1200 atcataaaaagaaataagaa aaagaggtgc gagcctcttt tttgtgtgac aaaataaaaa 1260 catctacctattcatatacg ctagtgtcat agtcctgaaa atcatctgca tcaagaacaa 1320 tttcacaactcttatacttt tctcttacaa gtcgttcggc ttcatctgga ttttcagcct 1380 ctatacttactaaacgtgat aaagtttctg taatttctac tgtatcgacc tgcagactgg 1440 ctgtgtataagggagcctga catttatatt ccccagaaca tcaggttaat ggcgtttttg 1500 atgtcattttcgcggtggct gagatcagcc acttcttccc cgataacgga gaccggcaca 1560 ctggccatatcggtggtcat catgcgccag ctttcatccc cgatatgcac caccgggtaa 1620 agttcacgggagactttatc tgacagcaga cgtgcactgg ccagggggat caccatccgt 1680 cgcccgggcgtgtcaataat atcactctgt acatccacaa acagacgata acggctctct 1740 cttttataggtgtaaacctt aaactgcatt tcaccagtcc ctgttctcgt cagcaaaaga 1800 gccgttcatttcaataaacc gggcgacctc agccatccct tcctgatttt ccgctttcca 1860 gcgttcggcacgcagacgac gggcttcatt ctgcatggtt gtgcttacca gaccggagat 1920 attgacatcatatatgcctt gagcaactga tagctgtcgc tgtcaactgt cactgtaata 1980 cgctgcttcatagcacacct ctttttgaca tacttcgggt atacatatca gtatatattc 2040 ttataccgcaaaaatcagcg cgcaaatacg catactgtta tctggctttt agtaagccgg 2100 atccacgcgattacgccccg ccctgccact catcgcagta ctgttgtaat tcattaagca 2160 ttctgccgacatggaagcca tcacagacgg catgatgaac ctgaatcgcc agcggcatca 2220 gcaccttgtcgccttgcgta taatatttgc ccatggtgaa aacgggggcg aagaagttgt 2280 ccatattggccacgtttaaa tcaaaactgg tgaaactcac ccagggattg gctgagacga 2340 aaaacatattctcaataaac cctttaggga aataggccag gttttcaccg taacacgcca 2400 catcttgcgaatatatgtgt agaaactgcc ggaaatcgtc gtggtattca ctccagagcg 2460 atgaaaacgtttcagtttgc tcatggaaaa cggtgtaaca agggtgaaca ctatcccata 2520 tcaccagctcaccgtctttc attgccatac ggaattccgg atgagcattc atcaggcggg 2580 caagaatgtgaataaaggcc ggataaaact tgtgcttatt tttctttacg gtctttaaaa 2640 aggccgtaatatccagctga acggtctggt tataggtaca ttgagcaact gactgaaatg 2700 cctcaaaatgttctttacga tgccattggg atatatcaac ggtggtatat ccagtgattt 2760 ttttctccattttagcttcc ttagctcctg aaaatctcga taactcaaaa aatacgcccg 2820 gtagtgatcttatttcatta tggtgaaagt tggaacctct tacgtgccga tcaacgtctc 2880 attttcgccaaaagttggcc cagggcttcc cggtatcaac agggacacca ggatttattt 2940 attctgcgaagtgatcttcc gtcacaggta tttattcggc gcaaagtgcg tcgggtgatg 3000 ctgccaacttagtcgactac aggtcactaa taccatctaa gtagttgatt catagtgact 3060 ggatatgttgtgttttacag tattatgtag tctgtttttt atgcaaaatc taatttaata 3120 tattgatatttatatcattt tacgtttctc gttcagcttt cttgtacaaa gtggttgatc 3180 tagagggcccgcggttcgaa ggtaagccta tccctaaccc tctcctcggt ctcgattcta 3240 cgcgtaccggttagtaatga gtttaaacgg gggaggctaa ctgaaacacg gaaggagaca 3300 ataccggaaggaacccgcgc tatgacggca ataaaaagac agaataaaac gcacgggtgt 3360 tgggtcgtttgttcataaac gcggggttcg gtcccagggc tggcactctg tcgatacccc 3420 accgagaccccattggggcc aatacgcccg cgtttcttcc ttttccccac cccacccccc 3480 aagttcgggtgaaggcccag ggctcgcagc caacgtcggg gcggcaggcc ctgccatagc 3540 agatctgcgcagctggggct ctagggggta tccccacgcg ccctgtagcg gcgcattaag 3600 cgcggcgggtgtggtggtta cgcgcagcgt gaccgctaca cttgccagcg ccctagcgcc 3660 cgctcctttcgctttcttcc cttcctttct cgccacgttc gccggctttc cccgtcaagc 3720 tctaaatcggggcatccctt tagggttccg atttagtgct ttacggcacc tcgaccccaa 3780 aaaacttgattagggtgatg gttcacgtag tgggccatcg ccctgataga cggtttttcg 3840 ccctttgacgttggagtcca cgttctttaa tagtggactc ttgttccaaa ctggaacaac 3900 actcaaccctatctcggtct attcttttga tttataaggg attttgggga tttcggccta 3960 ttggttaaaaaatgagctga tttaacaaaa atttaacgcg aattaattct gtggaatgtg 4020 tgtcagttagggtgtggaaa gtccccaggc tccccagcag gcagaagtat gcaaagcatg 4080 catctcaattagtcagcaac caggtgtgga aagtccccag gctccccagc aggcagaagt 4140 atgcaaagcatgcatctcaa ttagtcagca accatagtcc cgcccctaac tccgcccatc 4200 ccgcccctaactccgcccag ttccgcccat tctccgcccc atggctgact aatttttttt 4260 atttatgcagaggccgaggc cgcctctgcc tctgagctat tccagaagta gtgaggaggc 4320 ttttttggaggcctaggctt ttgcaaaaag ctcccgggag cttgtatatc cattttcgga 4380 tctgatcagcacgtgttgac aattaatcat cggcatagta tatcggcata gtataatacg 4440 acaaggtgaggaactaaacc atggccaagc ctttgtctca agaagaatcc accctcattg 4500 aaagagcaacggctacaatc aacagcatcc ccatctctga agactacagc gtcgccagcg 4560 cagctctctctagcgacggc cgcatcttca ctggtgtcaa tgtatatcat tttactgggg 4620 gaccttgtgcagaactcgtg gtgctgggca ctgctgctgc tgcggcagct ggcaacctga 4680 cttgtatcgtcgcgatcgga aatgagaaca ggggcatctt gagcccctgc ggacggtgcc 4740 gacaggtgcttctcgatctg catcctggga tcaaagccat agtgaaggac agtgatggac 4800 agccgacggcagttgggatt cgtgaattgc tgccctctgg ttatgtgtgg gagggctaag 4860 cacttcgtggccgaggagca ggactgacac gtgctacgag atttcgattc caccgccgcc 4920 ttctatgaaaggttgggctt cggaatcgtt ttccgggacg ccggctggat gatcctccag 4980 cgcggggatctcatgctgga gttcttcgcc caccccaact tgtttattgc agcttataat 5040 ggttacaaataaagcaatag catcacaaat ttcacaaata aagcattttt ttcactgcat 5100 tctagttgtggtttgtccaa actcatcaat gtatcttatc atgtctgtat accgtcgacc 5160 tctagctagagcttggcgta atcatggtca tagctgtttc ctgtgtgaaa ttgttatccg 5220 ctcacaattccacacaacat acgagccgga agcataaagt gtaaagcctg gggtgcctaa 5280 tgagtgagctaactcacatt aattgcgttg cgctcactgc ccgctttcca gtcgggaaac 5340 ctgtcgtgccagctgcatta atgaatcggc caacgcgcgg ggagaggcgg tttgcgtatt 5400 gggcgctcttccgcttcctc gctcactgac tcgctgcgct cggtcgttcg gctgcggcga 5460 gcggtatcagctcactcaaa ggcggtaata cggttatcca cagaatcagg ggataacgca 5520 ggaaagaacatgtgagcaaa aggccagcaa aaggccagga accgtaaaaa ggccgcgttg 5580 ctggcgtttttccataggct ccgcccccct gacgagcatc acaaaaatcg acgctcaagt 5640 cagaggtggcgaaacccgac aggactataa agataccagg cgtttccccc tggaagctcc 5700 ctcgtgcgctctcctgttcc gaccctgccg cttaccggat acctgtccgc ctttctccct 5760 tcgggaagcgtggcgctttc tcatagctca cgctgtaggt atctcagttc ggtgtaggtc 5820 gttcgctccaagctgggctg tgtgcacgaa ccccccgttc agcccgaccg ctgcgcctta 5880 tccggtaactatcgtcttga gtccaacccg gtaagacacg acttatcgcc actggcagca 5940 gccactggtaacaggattag cagagcgagg tatgtaggcg gtgctacaga gttcttgaag 6000 tggtggcctaactacggcta cactagaaga acagtatttg gtatctgcgc tctgctgaag 6060 ccagttaccttcggaaaaag agttggtagc tcttgatccg gcaaacaaac caccgctggt 6120 agcggtttttttgtttgcaa gcagcagatt acgcgcagaa aaaaaggatc tcaagaagat 6180 cctttgatcttttctacggg gtctgacgct cagtggaacg aaaactcacg ttaagggatt 6240 ttggtcatgagattatcaaa aaggatcttc acctagatcc ttttaaatta aaaatgaagt 6300 tttaaatcaatctaaagtat atatgagtaa acttggtctg acagttacca atgcttaatc 6360 agtgaggcacctatctcagc gatctgtcta tttcgttcat ccatagttgc ctgactcccc 6420 gtcgtgtagataactacgat acgggagggc ttaccatctg gccccagtgc tgcaatgata 6480 ccgcgagacccacgctcacc ggctccagat ttatcagcaa taaaccagcc agccggaagg 6540 gccgagcgcagaagtggtcc tgcaacttta tccgcctcca tccagtctat taattgttgc 6600 cgggaagctagagtaagtag ttcgccagtt aatagtttgc gcaacgttgt tgccattgct 6660 acaggcatcgtggtgtcacg ctcgtcgttt ggtatggctt cattcagctc cggttcccaa 6720 cgatcaaggcgagttacatg atcccccatg ttgtgcaaaa aagcggttag ctccttcggt 6780 cctccgatcgttgtcagaag taagttggcc gcagtgttat cactcatggt tatggcagca 6840 ctgcataattctcttactgt catgccatcc gtaagatgct tttctgtgac tggtgagtac 6900 tcaaccaagtcattctgaga atagtgtatg cggcgaccga gttgctcttg cccggcgtca 6960 atacgggataataccgcgcc acatagcaga actttaaaag tgctcatcat tggaaaacgt 7020 tcttcggggcgaaaactctc aaggatctta ccgctgttga gatccagttc gatgtaaccc 7080 actcgtgcacccaactgatc ttcagcatct tttactttca ccagcgtttc tgggtgagca 7140 aaaacaggaaggcaaaatgc cgcaaaaaag ggaataaggg cgacacggaa atgttgaata 7200 ctcatactcttcctttttca atattattga agcatttatc agggttattg tctcatgagc 7260 ggatacatatttgaatgtat ttagaaaaat aaacaaatag gggttccgcg cacatttccc 7320 cgaaaagtgccacctgacgt c 7341 113 7995 DNA Artificial Sequence pcDNA6.2/GFP-DEST 113gacggatcgg gagatctccc gatcccctat ggtgcactct cagtacaatc tgctctgatg 60ccgcatagtt aagccagtat ctgctccctg cttgtgtgtt ggaggtcgct gagtagtgcg 120cgagcaaaat ttaagctaca acaaggcaag gcttgaccga caattgcatg aagaatctgc 180ttagggttag gcgttttgcg ctgcttcgcg atgtacgggc cagatatacg cgttgacatt 240gattattgac tagttattaa tagtaatcaa ttacggggtc attagttcat agcccatata 300tggagttccg cgttacataa cttacggtaa atggcccgcc tggctgaccg cccaacgacc 360cccgcccatt gacgtcaata atgacgtatg ttcccatagt aacgccaata gggactttcc 420attgacgtca atgggtggag tatttacggt aaactgccca cttggcagta catcaagtgt 480atcatatgcc aagtacgccc cctattgacg tcaatgacgg taaatggccc gcctggcatt 540atgcccagta catgacctta tgggactttc ctacttggca gtacatctac gtattagtca 600tcgctattac catggtgatg cggttttggc agtacatcaa tgggcgtgga tagcggtttg 660actcacgggg atttccaagt ctccacccca ttgacgtcaa tgggagtttg ttttggcacc 720aaaatcaacg ggactttcca aaatgtcgta acaactccgc cccattgacg caaatgggcg 780gtaggcgtgt acggtgggag gtctatataa gcagagctct ctggctaact agagaaccca 840ctgcttactg gcttatcgaa attaatacga ctcactatag ggagacccaa gctggctagt 900taagctatca acaagtttgt acaaaaaagc tgaacgagaa acgtaaaatg atataaatat 960caatatatta aattagattt tgcataaaaa acagactaca taatactgta aaacacaaca 1020tatccagtca ctatgaatca actacttaga tggtattagt gacctgtagt cgaccgacag 1080ccttccaaat gttcttcggg tgatgctgcc aacttagtcg accgacagcc ttccaaatgt 1140tcttctcaaa cggaatcgtc gtatccagcc tactcgctat tgtcctcaat gccgtattaa 1200atcataaaaa gaaataagaa aaagaggtgc gagcctcttt tttgtgtgac aaaataaaaa 1260catctaccta ttcatatacg ctagtgtcat agtcctgaaa atcatctgca tcaagaacaa 1320tttcacaact cttatacttt tctcttacaa gtcgttcggc ttcatctgga ttttcagcct 1380ctatacttac taaacgtgat aaagtttctg taatttctac tgtatcgacc tgcagactgg 1440ctgtgtataa gggagcctga catttatatt ccccagaaca tcaggttaat ggcgtttttg 1500atgtcatttt cgcggtggct gagatcagcc acttcttccc cgataacgga gaccggcaca 1560ctggccatat cggtggtcat catgcgccag ctttcatccc cgatatgcac caccgggtaa 1620agttcacggg agactttatc tgacagcaga cgtgcactgg ccagggggat caccatccgt 1680cgcccgggcg tgtcaataat atcactctgt acatccacaa acagacgata acggctctct 1740cttttatagg tgtaaacctt aaactgcatt tcaccagtcc ctgttctcgt cagcaaaaga 1800gccgttcatt tcaataaacc gggcgacctc agccatccct tcctgatttt ccgctttcca 1860gcgttcggca cgcagacgac gggcttcatt ctgcatggtt gtgcttacca gaccggagat 1920attgacatca tatatgcctt gagcaactga tagctgtcgc tgtcaactgt cactgtaata 1980cgctgcttca tagcacacct ctttttgaca tacttcgggt atacatatca gtatatattc 2040ttataccgca aaaatcagcg cgcaaatacg catactgtta tctggctttt agtaagccgg 2100atccacgcga ttacgccccg ccctgccact catcgcagta ctgttgtaat tcattaagca 2160ttctgccgac atggaagcca tcacagacgg catgatgaac ctgaatcgcc agcggcatca 2220gcaccttgtc gccttgcgta taatatttgc ccatggtgaa aacgggggcg aagaagttgt 2280ccatattggc cacgtttaaa tcaaaactgg tgaaactcac ccagggattg gctgagacga 2340aaaacatatt ctcaataaac cctttaggga aataggccag gttttcaccg taacacgcca 2400catcttgcga atatatgtgt agaaactgcc ggaaatcgtc gtggtattca ctccagagcg 2460atgaaaacgt ttcagtttgc tcatggaaaa cggtgtaaca agggtgaaca ctatcccata 2520tcaccagctc accgtctttc attgccatac ggaattccgg atgagcattc atcaggcggg 2580caagaatgtg aataaaggcc ggataaaact tgtgcttatt tttctttacg gtctttaaaa 2640aggccgtaat atccagctga acggtctggt tataggtaca ttgagcaact gactgaaatg 2700cctcaaaatg ttctttacga tgccattggg atatatcaac ggtggtatat ccagtgattt 2760ttttctccat tttagcttcc ttagctcctg aaaatctcga taactcaaaa aatacgcccg 2820gtagtgatct tatttcatta tggtgaaagt tggaacctct tacgtgccga tcaacgtctc 2880attttcgcca aaagttggcc cagggcttcc cggtatcaac agggacacca ggatttattt 2940attctgcgaa gtgatcttcc gtcacaggta tttattcggc gcaaagtgcg tcgggtgatg 3000ctgccaactt agtcgactac aggtcactaa taccatctaa gtagttgatt catagtgact 3060ggatatgttg tgttttacag tattatgtag tctgtttttt atgcaaaatc taatttaata 3120tattgatatt tatatcattt tacgtttctc gttcagcttt cttgtacaaa gtggttgatc 3180tagagggccc cgcggctagc aaaggagaag aacttttcac tggagttgtc ccaattcttg 3240ttgaattaga tggtgatgtt aatgggcaca aattttctgt cagtggagag ggtgaaggtg 3300atgctacata cggaaagctt acccttaaat ttatttgcac tactggaaaa ctacctgttc 3360catggccaac acttgtcact actttctctt atggtgttca atgcttttcc cgttatccgg 3420atcatatgaa acggcatgac tttttcaaga gtgccatgcc cgaaggttat gtacaggaac 3480gcactatatc tttcaaagat gacgggaact acaagacgcg tgctgaagtc aagtttgaag 3540gtgataccct tgttaatcgt atcgagttaa aaggtattga ttttaaagaa gatggaaaca 3600ttctcggaca caaactcgag tacaactata actcacacaa tgtatacatc acggcagaca 3660aacaaaagaa tggaatcaaa gctaacttca aaattcgtca caacattgaa gatggatccg 3720ttcaactagc agaccattat caacaaaata ctccaattgg cgatggccct gtccttttac 3780cagacaacca ttacctgtcg acacaatctg ccctttcgaa agatcccaac gaaaagcgtg 3840accacatggt ccttcttgag tttgtaactg ctgctgggat tacacatggc atggatgaat 3900agtaatgagt ccacgtttaa acgggggagg ctaactgaaa cacggaagga gacaataccg 3960gaaggaaccc gcgctatgac ggcaataaaa agacagaata aaacgcacgg gtgttgggtc 4020gtttgttcat aaacgcgggg ttcggtccca gggctggcac tctgtcgata ccccaccgag 4080accccattgg ggccaatacg cccgcgtttc ttccttttcc ccaccccacc ccccaagttc 4140gggtgaaggc ccagggctcg cagccaacgt cggggcggca ggccctgcca tagcagatct 4200gcgcagctgg ggctctaggg ggtatcccca cgcgccctgt agcggcgcat taagcgcggc 4260gggtgtggtg gttacgcgca gcgtgaccgc tacacttgcc agcgccctag cgcccgctcc 4320tttcgctttc ttcccttcct ttctcgccac gttcgccggc tttccccgtc aagctctaaa 4380tcggggcatc cctttagggt tccgatttag tgctttacgg cacctcgacc ccaaaaaact 4440tgattagggt gatggttcac gtagtgggcc atcgccctga tagacggttt ttcgcccttt 4500gacgttggag tccacgttct ttaatagtgg actcttgttc caaactggaa caacactcaa 4560ccctatctcg gtctattctt ttgatttata agggattttg gggatttcgg cctattggtt 4620aaaaaatgag ctgatttaac aaaaatttaa cgcgaattaa ttctgtggaa tgtgtgtcag 4680ttagggtgtg gaaagtcccc aggctcccca gcaggcagaa gtatgcaaag catgcatctc 4740aattagtcag caaccaggtg tggaaagtcc ccaggctccc cagcaggcag aagtatgcaa 4800agcatgcatc tcaattagtc agcaaccata gtcccgcccc taactccgcc catcccgccc 4860ctaactccgc ccagttccgc ccattctccg ccccatggct gactaatttt ttttatttat 4920gcagaggccg aggccgcctc tgcctctgag ctattccaga agtagtgagg aggctttttt 4980ggaggcctag gcttttgcaa aaagctcccg ggagcttgta tatccatttt cggatctgat 5040cagcacgtgt tgacaattaa tcatcggcat agtatatcgg catagtataa tacgacaagg 5100tgaggaacta aaccatggcc aagcctttgt ctcaagaaga atccaccctc attgaaagag 5160caacggctac aatcaacagc atccccatct ctgaagacta cagcgtcgcc agcgcagctc 5220tctctagcga cggccgcatc ttcactggtg tcaatgtata tcattttact gggggacctt 5280gtgcagaact cgtggtgctg ggcactgctg ctgctgcggc agctggcaac ctgacttgta 5340tcgtcgcgat cggaaatgag aacaggggca tcttgagccc ctgcggacgg tgccgacagg 5400tgcttctcga tctgcatcct gggatcaaag ccatagtgaa ggacagtgat ggacagccga 5460cggcagttgg gattcgtgaa ttgctgccct ctggttatgt gtgggagggc taagcacttc 5520gtggccgagg agcaggactg acacgtgcta cgagatttcg attccaccgc cgccttctat 5580gaaaggttgg gcttcggaat cgttttccgg gacgccggct ggatgatcct ccagcgcggg 5640gatctcatgc tggagttctt cgcccacccc aacttgttta ttgcagctta taatggttac 5700aaataaagca atagcatcac aaatttcaca aataaagcat ttttttcact gcattctagt 5760tgtggtttgt ccaaactcat caatgtatct tatcatgtct gtataccgtc gacctctagc 5820tagagcttgg cgtaatcatg gtcatagctg tttcctgtgt gaaattgtta tccgctcaca 5880attccacaca acatacgagc cggaagcata aagtgtaaag cctggggtgc ctaatgagtg 5940agctaactca cattaattgc gttgcgctca ctgcccgctt tccagtcggg aaacctgtcg 6000tgccagctgc attaatgaat cggccaacgc gcggggagag gcggtttgcg tattgggcgc 6060tcttccgctt cctcgctcac tgactcgctg cgctcggtcg ttcggctgcg gcgagcggta 6120tcagctcact caaaggcggt aatacggtta tccacagaat caggggataa cgcaggaaag 6180aacatgtgag caaaaggcca gcaaaaggcc aggaaccgta aaaaggccgc gttgctggcg 6240tttttccata ggctccgccc ccctgacgag catcacaaaa atcgacgctc aagtcagagg 6300tggcgaaacc cgacaggact ataaagatac caggcgtttc cccctggaag ctccctcgtg 6360cgctctcctg ttccgaccct gccgcttacc ggatacctgt ccgcctttct cccttcggga 6420agcgtggcgc tttctcatag ctcacgctgt aggtatctca gttcggtgta ggtcgttcgc 6480tccaagctgg gctgtgtgca cgaacccccc gttcagcccg accgctgcgc cttatccggt 6540aactatcgtc ttgagtccaa cccggtaaga cacgacttat cgccactggc agcagccact 6600ggtaacagga ttagcagagc gaggtatgta ggcggtgcta cagagttctt gaagtggtgg 6660cctaactacg gctacactag aagaacagta tttggtatct gcgctctgct gaagccagtt 6720accttcggaa aaagagttgg tagctcttga tccggcaaac aaaccaccgc tggtagcggt 6780ttttttgttt gcaagcagca gattacgcgc agaaaaaaag gatctcaaga agatcctttg 6840atcttttcta cggggtctga cgctcagtgg aacgaaaact cacgttaagg gattttggtc 6900atgagattat caaaaaggat cttcacctag atccttttaa attaaaaatg aagttttaaa 6960tcaatctaaa gtatatatga gtaaacttgg tctgacagtt accaatgctt aatcagtgag 7020gcacctatct cagcgatctg tctatttcgt tcatccatag ttgcctgact ccccgtcgtg 7080tagataacta cgatacggga gggcttacca tctggcccca gtgctgcaat gataccgcga 7140gacccacgct caccggctcc agatttatca gcaataaacc agccagccgg aagggccgag 7200cgcagaagtg gtcctgcaac tttatccgcc tccatccagt ctattaattg ttgccgggaa 7260gctagagtaa gtagttcgcc agttaatagt ttgcgcaacg ttgttgccat tgctacaggc 7320atcgtggtgt cacgctcgtc gtttggtatg gcttcattca gctccggttc ccaacgatca 7380aggcgagtta catgatcccc catgttgtgc aaaaaagcgg ttagctcctt cggtcctccg 7440atcgttgtca gaagtaagtt ggccgcagtg ttatcactca tggttatggc agcactgcat 7500aattctctta ctgtcatgcc atccgtaaga tgcttttctg tgactggtga gtactcaacc 7560aagtcattct gagaatagtg tatgcggcga ccgagttgct cttgcccggc gtcaatacgg 7620gataataccg cgccacatag cagaacttta aaagtgctca tcattggaaa acgttcttcg 7680gggcgaaaac tctcaaggat cttaccgctg ttgagatcca gttcgatgta acccactcgt 7740gcacccaact gatcttcagc atcttttact ttcaccagcg tttctgggtg agcaaaaaca 7800ggaaggcaaa atgccgcaaa aaagggaata agggcgacac ggaaatgttg aatactcata 7860ctcttccttt ttcaatatta ttgaagcatt tatcagggtt attgtctcat gagcggatac 7920atatttgaat gtatttagaa aaataaacaa ataggggttc cgcgcacatt tccccgaaaa 7980gtgccacctg acgtc 7995 114 265 PRT Unknown Amino acid sequence of apolypeptide having beta-lactamase activity 114 Met Gly His Pro Glu ThrLeu Val Lys Val Lys Asp Ala Glu Asp Gln 1 5 10 15 Leu Gly Ala Arg ValGly Tyr Ile Glu Leu Asp Leu Asn Ser Gly Lys 20 25 30 Ile Leu Glu Ser PheArg Pro Glu Glu Arg Phe Pro Met Met Ser Thr 35 40 45 Phe Lys Val Leu LeuCys Gly Ala Val Leu Ser Arg Asp Asp Ala Gly 50 55 60 Gln Glu Gln Leu GlyArg Arg Ile His Tyr Ser Gln Asn Asp Leu Val 65 70 75 80 Glu Tyr Ser ProVal Thr Glu Lys His Leu Thr Asp Gly Met Thr Val 85 90 95 Arg Glu Leu CysSer Ala Ala Ile Thr Met Ser Asp Asn Thr Ala Ala 100 105 110 Asn Leu LeuLeu Thr Thr Ile Gly Gly Pro Lys Glu Leu Thr Ala Phe 115 120 125 Leu HisAsn Met Gly Asp His Val Thr Arg Leu Asp His Trp Glu Pro 130 135 140 GluLeu Asn Glu Ala Ile Pro Asn Asp Glu Arg Asp Thr Thr Met Pro 145 150 155160 Val Ala Met Ala Thr Thr Leu Arg Lys Leu Leu Thr Gly Glu Leu Leu 165170 175 Thr Leu Ala Ser Arg Gln Gln Leu Ile Asp Trp Met Glu Ala Asp Lys180 185 190 Val Ala Gly Pro Leu Leu Arg Ser Ala Leu Pro Ala Gly Trp PheIle 195 200 205 Ala Asp Lys Ser Gly Ala Gly Glu Arg Gly Ser Arg Gly IleIle Ala 210 215 220 Ala Leu Gly Pro Asp Gly Lys Pro Ser Arg Ile Val ValIle Tyr Thr 225 230 235 240 Thr Gly Ser Gln Ala Thr Met Asp Glu Arg AsnArg Gln Ile Ala Glu 245 250 255 Ile Gly Ala Ser Leu Ile Lys His Trp 260265 115 8599 DNA Artificial Sequence pLenti4TO/V5-DEST 115 aatgtagtcttatgcaatac tcttgtagtc ttgcaacatg gtaacgatga gttagcaaca 60 tgccttacaaggagagaaaa agcaccgtgc atgccgattg gtggaagtaa ggtggtacga 120 tcgtgccttattaggaaggc aacagacggg tctgacatgg attggacgaa ccactgaatt 180 gccgcattgcagagatattg tatttaagtg cctagctcga tacataaacg ggtctctctg 240 gttagaccagatctgagcct gggagctctc tggctaacta gggaacccac tgcttaagcc 300 tcaataaagcttgccttgag tgcttcaagt agtgtgtgcc cgtctgttgt gtgactctgg 360 taactagagatccctcagac ccttttagtc agtgtggaaa atctctagca gtggcgcccg 420 aacagggacttgaaagcgaa agggaaacca gaggagctct ctcgacgcag gactcggctt 480 gctgaagcgcgcacggcaag aggcgagggg cggcgactgg tgagtacgcc aaaaattttg 540 actagcggaggctagaagga gagagatggg tgcgagagcg tcagtattaa gcgggggaga 600 attagatcgcgatgggaaaa aattcggtta aggccagggg gaaagaaaaa atataaatta 660 aaacatatagtatgggcaag cagggagcta gaacgattcg cagttaatcc tggcctgtta 720 gaaacatcagaaggctgtag acaaatactg ggacagctac aaccatccct tcagacagga 780 tcagaagaacttagatcatt atataataca gtagcaaccc tctattgtgt gcatcaaagg 840 atagagataaaagacaccaa ggaagcttta gacaagatag aggaagagca aaacaaaagt 900 aagaccaccgcacagcaagc ggccgctgat cttcagacct ggaggaggag atatgaggga 960 caattggagaagtgaattat ataaatataa agtagtaaaa attgaaccat taggagtagc 1020 acccaccaaggcaaagagaa gagtggtgca gagagaaaaa agagcagtgg gaataggagc 1080 tttgttccttgggttcttgg gagcagcagg aagcactatg ggcgcagcgt caatgacgct 1140 gacggtacaggccagacaat tattgtctgg tatagtgcag cagcagaaca atttgctgag 1200 ggctattgaggcgcaacagc atctgttgca actcacagtc tggggcatca agcagctcca 1260 ggcaagaatcctggctgtgg aaagatacct aaaggatcaa cagctcctgg ggatttgggg 1320 ttgctctggaaaactcattt gcaccactgc tgtgccttgg aatgctagtt ggagtaataa 1380 atctctggaacagatttgga atcacacgac ctggatggag tgggacagag aaattaacaa 1440 ttacacaagcttaatacact ccttaattga agaatcgcaa aaccagcaag aaaagaatga 1500 acaagaattattggaattag ataaatgggc aagtttgtgg aattggttta acataacaaa 1560 ttggctgtggtatataaaat tattcataat gatagtagga ggcttggtag gtttaagaat 1620 agtttttgctgtactttcta tagtgaatag agttaggcag ggatattcac cattatcgtt 1680 tcagacccacctcccaaccc cgaggggacc cgacaggccc gaaggaatag aagaagaagg 1740 tggagagagagacagagaca gatccattcg attagtgaac ggatctcgac ggtatcgata 1800 agcttgggagttccgcgtta cataacttac ggtaaatggc ccgcctggct gaccgcccaa 1860 cgacccccgcccattgacgt caataatgac gtatgttccc atagtaacgc caatagggac 1920 tttccattgacgtcaatggg tggagtattt acggtaaact gcccacttgg cagtacatca 1980 agtgtatcatatgccaagta cgccccctat tgacgtcaat gacggtaaat ggcccgcctg 2040 gcattatgcccagtacatga ccttatggga ctttcctact tggcagtaca tctacgtatt 2100 agtcatcgctattaccatgg tgatgcggtt ttggcagtac atcaatgggc gtggatagcg 2160 gtttgactcacggggatttc caagtctcca ccccattgac gtcaatggga gtttgttttg 2220 gcaccaaaatcaacgggact ttccaaaatg tcgtaacaac tccgccccat tgacgcaaat 2280 gggcggtaggcgtgtacggt gggaggtcta tataagcaga gctctcccta tcagtgatag 2340 agatctccctatcagtgata gagatcgtcg actagtccag tgtggtggaa ttctgcagat 2400 atcaacaagtttgtacaaaa aagctgaacg agaaacgtaa aatgatataa atatcaatat 2460 attaaattagattttgcata aaaaacagac tacataatac tgtaaaacac aacatatcca 2520 gtcactatggcggccgcatt aggcacccca ggctttacac tttatgcttc cggctcgtat 2580 aatgtgtggattttgagtta ggatccggcg agattttcag gagctaagga agctaaaatg 2640 gagaaaaaaatcactggata taccaccgtt gatatatccc aatggcatcg taaagaacat 2700 tttgaggcatttcagtcagt tgctcaatgt acctataacc agaccgttca gctggatatt 2760 acggcctttttaaagaccgt aaagaaaaat aagcacaagt tttatccggc ctttattcac 2820 attcttgcccgcctgatgaa tgctcatccg gaattccgta tggcaatgaa agacggtgag 2880 ctggtgatatgggatagtgt tcacccttgt tacaccgttt tccatgagca aactgaaacg 2940 ttttcatcgctctggagtga ataccacgac gatttccggc agtttctaca catatattcg 3000 caagatgtggcgtgttacgg tgaaaacctg gcctatttcc ctaaagggtt tattgagaat 3060 atgtttttcgtctcagccaa tccctgggtg agtttcacca gttttgattt aaacgtggcc 3120 aatatggacaacttcttcgc ccccgttttc accatgggca aatattatac gcaaggcgac 3180 aaggtgctgatgccgctggc gattcaggtt catcatgccg tctgtgatgg cttccatgtc 3240 ggcagaatgcttaatgaatt acaacagtac tgcgatgagt ggcagggcgg ggcgtaaaga 3300 tctggatccggcttactaaa agccagataa cagtatgcgt atttgcgcgc tgatttttgc 3360 ggtataagaatatatactga tatgtatacc cgaagtatgt caaaaagagg tgtgctatga 3420 agcagcgtattacagtgaca gttgacagcg acagctatca gttgctcaag gcatatatga 3480 tgtcaatatctccggtctgg taagcacaac catgcagaat gaagcccgtc gtctgcgtgc 3540 cgaacgctggaaagcggaaa atcaggaagg gatggctgag gtcgcccggt ttattgaaat 3600 gaacggctcttttgctgacg agaacaggga ctggtgaaat gcagtttaag gtttacacct 3660 ataaaagagagagccgttat cgtctgtttg tggatgtaca gagtgatatt attgacacgc 3720 ccgggcgacggatggtgatc cccctggcca gtgcacgtct gctgtcagat aaagtctccc 3780 gtgaactttacccggtggtg catatcgggg atgaaagctg gcgcatgatg accaccgata 3840 tggccagtgtgccggtctcc gttatcgggg aagaagtggc tgatctcagc caccgcgaaa 3900 atgacatcaaaaacgccatt aacctgatgt tctggggaat ataaatgtca ggctccgtta 3960 tacacagccagtctgcaggt cgaccatagt gactggatat gttgtgtttt acagtattat 4020 gtagtctgttttttatgcaa aatctaattt aatatattga tatttatatc attttacgtt 4080 tctcgttcagctttcttgta caaagtggtt gatatccagc acagtggcgg ccgctcgagt 4140 ctagagggcccgcggttcga aggtaagcct atccctaacc ctctcctcgg tctcgattct 4200 acgcgtaccggttagtaatg agtttggaat taattctgtg gaatgtgtgt cagttagggt 4260 gtggaaagtccccaggctcc ccaggcaggc agaagtatgc aaagcatgca tctcaattag 4320 tcagcaaccaggtgtggaaa gtccccaggc tccccagcag gcagaagtat gcaaagcatg 4380 catctcaattagtcagcaac catagtcccg cccctaactc cgcccatccc gcccctaact 4440 ccgcccagttccgcccattc tccgccccat ggctgactaa ttttttttat ttatgcagag 4500 gccgaggccgcctctgcctc tgagctattc cagaagtagt gaggaggctt ttttggaggc 4560 ctaggcttttgcaaaaagct ccccctgttg acaattaatc atcggcatag tatatcggca 4620 tagtataatacgacaaggtg aggaactaaa ccatggccaa gttgaccagt gccgttccgg 4680 tgctcaccgcgcgcgacgtc gccggagcgg tcgagttctg gaccgaccgg ctcgggttct 4740 cccgggacttcgtggaggac gacttcgccg gtgtggtccg ggacgacgtg accctgttca 4800 tcagcgcggtccaggaccag gtggtgccgg acaacaccct ggcctgggtg tgggtgcgcg 4860 gcctggacgagctgtacgcc gagtggtcgg aggtcgtgtc cacgaacttc cgggacgcct 4920 ccgggccggccatgaccgag atcggcgagc agccgtgggg gcgggagttc gccctgcgcg 4980 acccggccggcaactgcgtg cacttcgtgg ccgaggagca ggactgacac gtgctacgag 5040 atttaaatggtacctttaag accaatgact tacaaggcag ctgtagatct tagccacttt 5100 ttaaaagaaaaggggggact ggaagggcta attcactccc aacgaagaca agatctgctt 5160 tttgcttgtactgggtctct ctggttagac cagatctgag cctgggagct ctctggctaa 5220 ctagggaacccactgcttaa gcctcaataa agcttgcctt gagtgcttca agtagtgtgt 5280 gcccgtctgttgtgtgactc tggtaactag agatccctca gaccctttta gtcagtgtgg 5340 aaaatctctagcagtagtag ttcatgtcat cttattattc agtatttata acttgcaaag 5400 aaatgaatatcagagagtga gaggaacttg tttattgcag cttataatgg ttacaaataa 5460 agcaatagcatcacaaattt cacaaataaa gcattttttt cactgcattc tagttgtggt 5520 ttgtccaaactcatcaatgt atcttatcat gtctggctct agctatcccg cccctaactc 5580 cgcccatcccgcccctaact ccgcccagtt ccgcccattc tccgccccat ggctgactaa 5640 ttttttttatttatgcagag gccgaggccg cctcggcctc tgagctattc cagaagtagt 5700 gaggaggcttttttggaggc ctagggacgt acccaattcg ccctatagtg agtcgtatta 5760 cgcgcgctcactggccgtcg ttttacaacg tcgtgactgg gaaaaccctg gcgttaccca 5820 acttaatcgccttgcagcac atcccccttt cgccagctgg cgtaatagcg aagaggcccg 5880 caccgatcgcccttcccaac agttgcgcag cctgaatggc gaatgggacg cgccctgtag 5940 cggcgcattaagcgcggcgg gtgtggtggt tacgcgcagc gtgaccgcta cacttgccag 6000 cgccctagcgcccgctcctt tcgctttctt cccttccttt ctcgccacgt tcgccggctt 6060 tccccgtcaagctctaaatc gggggctccc tttagggttc cgatttagtg ctttacggca 6120 cctcgaccccaaaaaacttg attagggtga tggttcacgt agtgggccat cgccctgata 6180 gacggtttttcgccctttga cgttggagtc cacgttcttt aatagtggac tcttgttcca 6240 aactggaacaacactcaacc ctatctcggt ctattctttt gatttataag ggattttgcc 6300 gatttcggcctattggttaa aaaatgagct gatttaacaa aaatttaacg cgaattttaa 6360 caaaatattaacgcttacaa tttaggtggc acttttcggg gaaatgtgcg cggaacccct 6420 atttgtttatttttctaaat acattcaaat atgtatccgc tcatgagaca ataaccctga 6480 taaatgcttcaataatattg aaaaaggaag agtatgagta ttcaacattt ccgtgtcgcc 6540 cttattcccttttttgcggc attttgcctt cctgtttttg ctcacccaga aacgctggtg 6600 aaagtaaaagatgctgaaga tcagttgggt gcacgagtgg gttacatcga actggatctc 6660 aacagcggtaagatccttga gagttttcgc cccgaagaac gttttccaat gatgagcact 6720 tttaaagttctgctatgtgg cgcggtatta tcccgtattg acgccgggca agagcaactc 6780 ggtcgccgcatacactattc tcagaatgac ttggttgagt actcaccagt cacagaaaag 6840 catcttacggatggcatgac agtaagagaa ttatgcagtg ctgccataac catgagtgat 6900 aacactgcggccaacttact tctgacaacg atcggaggac cgaaggagct aaccgctttt 6960 ttgcacaacatgggggatca tgtaactcgc cttgatcgtt gggaaccgga gctgaatgaa 7020 gccataccaaacgacgagcg tgacaccacg atgcctgtag caatggcaac aacgttgcgc 7080 aaactattaactggcgaact acttactcta gcttcccggc aacaattaat agactggatg 7140 gaggcggataaagttgcagg accacttctg cgctcggccc ttccggctgg ctggtttatt 7200 gctgataaatctggagccgg tgagcgtggg tctcgcggta tcattgcagc actggggcca 7260 gatggtaagccctcccgtat cgtagttatc tacacgacgg ggagtcaggc aactatggat 7320 gaacgaaatagacagatcgc tgagataggt gcctcactga ttaagcattg gtaactgtca 7380 gaccaagtttactcatatat actttagatt gatttaaaac ttcattttta atttaaaagg 7440 atctaggtgaagatcctttt tgataatctc atgaccaaaa tcccttaacg tgagttttcg 7500 ttccactgagcgtcagaccc cgtagaaaag atcaaaggat cttcttgaga tccttttttt 7560 ctgcgcgtaatctgctgctt gcaaacaaaa aaaccaccgc taccagcggt ggtttgtttg 7620 ccggatcaagagctaccaac tctttttccg aaggtaactg gcttcagcag agcgcagata 7680 ccaaatactgttcttctagt gtagccgtag ttaggccacc acttcaagaa ctctgtagca 7740 ccgcctacatacctcgctct gctaatcctg ttaccagtgg ctgctgccag tggcgataag 7800 tcgtgtcttaccgggttgga ctcaagacga tagttaccgg ataaggcgca gcggtcgggc 7860 tgaacggggggttcgtgcac acagcccagc ttggagcgaa cgacctacac cgaactgaga 7920 tacctacagcgtgagctatg agaaagcgcc acgcttcccg aagggagaaa ggcggacagg 7980 tatccggtaagcggcagggt cggaacagga gagcgcacga gggagcttcc agggggaaac 8040 gcctggtatctttatagtcc tgtcgggttt cgccacctct gacttgagcg tcgatttttg 8100 tgatgctcgtcaggggggcg gagcctatgg aaaaacgcca gcaacgcggc ctttttacgg 8160 ttcctggccttttgctggcc ttttgctcac atgttctttc ctgcgttatc ccctgattct 8220 gtggataaccgtattaccgc ctttgagtga gctgataccg ctcgccgcag ccgaacgacc 8280 gagcgcagcgagtcagtgag cgaggaagcg gaagagcgcc caatacgcaa accgcctctc 8340 cccgcgcgttggccgattca ttaatgcagc tggcacgaca ggtttcccga ctggaaagcg 8400 ggcagtgagcgcaacgcaat taatgtgagt tagctcactc attaggcacc ccaggcttta 8460 cactttatgcttccggctcg tatgttgtgt ggaattgtga gcggataaca atttcacaca 8520 ggaaacagctatgaccatga ttacgccaag cgcgcaatta accctcacta aagggaacaa 8580 aagctggagctgcaagctt 8599 116 8355 DNA Artificial Sequence pLenti6/TR 116aatgtagtct tatgcaatac tcttgtagtc ttgcaacatg gtaacgatga gttagcaaca 60tgccttacaa ggagagaaaa agcaccgtgc atgccgattg gtggaagtaa ggtggtacga 120tcgtgcctta ttaggaaggc aacagacggg tctgacatgg attggacgaa ccactgaatt 180gccgcattgc agagatattg tatttaagtg cctagctcga tacataaacg ggtctctctg 240gttagaccag atctgagcct gggagctctc tggctaacta gggaacccac tgcttaagcc 300tcaataaagc ttgccttgag tgcttcaagt agtgtgtgcc cgtctgttgt gtgactctgg 360taactagaga tccctcagac ccttttagtc agtgtggaaa atctctagca gtggcgcccg 420aacagggact tgaaagcgaa agggaaacca gaggagctct ctcgacgcag gactcggctt 480gctgaagcgc gcacggcaag aggcgagggg cggcgactgg tgagtacgcc aaaaattttg 540actagcggag gctagaagga gagagatggg tgcgagagcg tcagtattaa gcgggggaga 600attagatcgc gatgggaaaa aattcggtta aggccagggg gaaagaaaaa atataaatta 660aaacatatag tatgggcaag cagggagcta gaacgattcg cagttaatcc tggcctgtta 720gaaacatcag aaggctgtag acaaatactg ggacagctac aaccatccct tcagacagga 780tcagaagaac ttagatcatt atataataca gtagcaaccc tctattgtgt gcatcaaagg 840atagagataa aagacaccaa ggaagcttta gacaagatag aggaagagca aaacaaaagt 900aagaccaccg cacagcaagc ggccgctgat cttcagacct ggaggaggag atatgaggga 960caattggaga agtgaattat ataaatataa agtagtaaaa attgaaccat taggagtagc 1020acccaccaag gcaaagagaa gagtggtgca gagagaaaaa agagcagtgg gaataggagc 1080tttgttcctt gggttcttgg gagcagcagg aagcactatg ggcgcagcgt caatgacgct 1140gacggtacag gccagacaat tattgtctgg tatagtgcag cagcagaaca atttgctgag 1200ggctattgag gcgcaacagc atctgttgca actcacagtc tggggcatca agcagctcca 1260ggcaagaatc ctggctgtgg aaagatacct aaaggatcaa cagctcctgg ggatttgggg 1320ttgctctgga aaactcattt gcaccactgc tgtgccttgg aatgctagtt ggagtaataa 1380atctctggaa cagatttgga atcacacgac ctggatggag tgggacagag aaattaacaa 1440ttacacaagc ttaatacact ccttaattga agaatcgcaa aaccagcaag aaaagaatga 1500acaagaatta ttggaattag ataaatgggc aagtttgtgg aattggttta acataacaaa 1560ttggctgtgg tatataaaat tattcataat gatagtagga ggcttggtag gtttaagaat 1620agtttttgct gtactttcta tagtgaatag agttaggcag ggatattcac cattatcgtt 1680tcagacccac ctcccaaccc cgaggggacc cgacaggccc gaaggaatag aagaagaagg 1740tggagagaga gacagagaca gatccattcg attagtgaac ggatctcgac ggtatcgata 1800agcttgggag ttccgcgtta cataacttac ggtaaatggc ccgcctggct gaccgcccaa 1860cgacccccgc ccattgacgt caataatgac gtatgttccc atagtaacgc caatagggac 1920tttccattga cgtcaatggg tggagtattt acggtaaact gcccacttgg cagtacatca 1980agtgtatcat atgccaagta cgccccctat tgacgtcaat gacggtaaat ggcccgcctg 2040gcattatgcc cagtacatga ccttatggga ctttcctact tggcagtaca tctacgtatt 2100agtcatcgct attaccatgg tgatgcggtt ttggcagtac atcaatgggc gtggatagcg 2160gtttgactca cggggatttc caagtctcca ccccattgac gtcaatggga gtttgttttg 2220gcaccaaaat caacgggact ttccaaaatg tcgtaacaac tccgccccat tgacgcaaat 2280gggcggtagg cgtgtacggt gggaggtcta tataagcaga gctcgtttag tgaaccgtca 2340gatcgcctgg agacgccatc cacgctgttt tgacctccat agaagacacc gactctagag 2400gatccactag tccagtgtgg tggaattctg cagatagctt ggtacccggg gatcctctag 2460ggcctctgag ctattccaga agtagtgaag aggctttttt ggaggcctag gcttttgcaa 2520aaagctccgg atcgatcctg agaacttcag ggtgagtttg gggacccttg attgttcttt 2580ctttttcgct attgtaaaat tcatgttata tggagggggc aaagttttca gggtgttgtt 2640tagaatggga agatgtccct tgtatcacca tggaccctca tgataatttt gtttctttca 2700ctttctactc tgttgacaac cattgtctcc tcttattttc ttttcatttt ctgtaacttt 2760ttcgttaaac tttagcttgc atttgtaacg aatttttaaa ttcacttttg tttatttgtc 2820agattgtaag tactttctct aatcactttt ttttcaaggc aatcagggta tattatattg 2880tacttcagca cagttttaga gaacaattgt tataattaaa tgataaggta gaatatttct 2940gcatataaat tctggctggc gtggaaatat tcttattggt agaaacaact acatcctggt 3000catcatcctg cctttctctt tatggttaca atgatataca ctgtttgaga tgaggataaa 3060atactctgag tccaaaccgg gcccctctgc taaccatgtt catgccttct tctttttcct 3120acagctcctg ggcaacgtgc tggttattgt gctgtctcat cattttggca aagaattgta 3180atacgactca ctatagggcg aattgatatg tctagattag ataaaagtaa agtgattaac 3240agcgcattag agctgcttaa tgaggtcgga atcgaaggtt taacaacccg taaactcgcc 3300cagaagctag gtgtagagca gcctacattg tattggcatg taaaaaataa gcgggctttg 3360ctcgacgcct tagccattga gatgttagat aggcaccata ctcacttttg ccctttagaa 3420ggggaaagct ggcaagattt tttacgtaat aacgctaaaa gttttagatg tgctttacta 3480agtcatcgcg atggagcaaa agtacattta ggtacacggc ctacagaaaa acagtatgaa 3540actctcgaaa atcaattagc ctttttatgc caacaaggtt tttcactaga gaatgcatta 3600tatgcactca gcgctgtggg gcattttact ttaggttgcg tattggaaga tcaagagcat 3660caagtcgcta aagaagaaag ggaaacacct actactgata gtatgccgcc attattacga 3720caagctatcg aattatttga tcaccaaggt gcagagccag ccttcttatt cggccttgaa 3780ttgatcatat gcggattaga aaaacaactt aaatgtgaaa gtgggtccgc gtacagcgga 3840tcccgggaat tctagagggc ccgcggttcg aacaaaaact catctcagaa gaggatctga 3900atatgcatac cggttagtaa tgagtttgga attaattctg tggaatgtgt gtcagttagg 3960gtgtggaaag tccccaggct ccccaggcag gcagaagtat gcaaagcatg catctcaatt 4020agtcagcaac caggtgtgga aagtccccag gctccccagc aggcagaagt atgcaaagca 4080tgcatctcaa ttagtcagca accatagtcc cgcccctaac tccgcccatc ccgcccctaa 4140ctccgcccag ttccgcccat tctccgcccc atggctgact aatttttttt atttatgcag 4200aggccgaggc cgcctctgcc tctgagctat tccagaagta gtgaggaggc ttttttggag 4260gcctaggctt ttgcaaaaag ctcccgggag cttgtatatc cattttcgga tctgatcagc 4320acgtgttgac aattaatcat cggcatagta tatcggcata gtataatacg acaaggtgag 4380gaactaaacc atggccaagc ctttgtctca agaagaatcc accctcattg aaagagcaac 4440ggctacaatc aacagcatcc ccatctctga agactacagc gtcgccagcg cagctctctc 4500tagcgacggc cgcatcttca ctggtgtcaa tgtatatcat tttactgggg gaccttgtgc 4560agaactcgtg gtgctgggca ctgctgctgc tgcggcagct ggcaacctga cttgtatcgt 4620cgcgatcgga aatgagaaca ggggcatctt gagcccctgc ggacggtgcc gacaggtgct 4680tctcgatctg catcctggga tcaaagccat agtgaaggac agtgatggac agccgacggc 4740agttgggatt cgtgaattgc tgccctctgg ttatgtgtgg gagggctaag cacaattcga 4800gctcggtacc tttaagacca atgacttaca aggcagctgt agatcttagc cactttttaa 4860aagaaaaggg gggactggaa gggctaattc actcccaacg aagacaagat ctgctttttg 4920cttgtactgg gtctctctgg ttagaccaga tctgagcctg ggagctctct ggctaactag 4980ggaacccact gcttaagcct caataaagct tgccttgagt gcttcaagta gtgtgtgccc 5040gtctgttgtg tgactctggt aactagagat ccctcagacc cttttagtca gtgtggaaaa 5100tctctagcag tagtagttca tgtcatctta ttattcagta tttataactt gcaaagaaat 5160gaatatcaga gagtgagagg aacttgttta ttgcagctta taatggttac aaataaagca 5220atagcatcac aaatttcaca aataaagcat ttttttcact gcattctagt tgtggtttgt 5280ccaaactcat caatgtatct tatcatgtct ggctctagct atcccgcccc taactccgcc 5340catcccgccc ctaactccgc ccagttccgc ccattctccg ccccatggct gactaatttt 5400ttttatttat gcagaggccg aggccgcctc ggcctctgag ctattccaga agtagtgagg 5460aggctttttt ggaggcctag ggacgtaccc aattcgccct atagtgagtc gtattacgcg 5520cgctcactgg ccgtcgtttt acaacgtcgt gactgggaaa accctggcgt tacccaactt 5580aatcgccttg cagcacatcc ccctttcgcc agctggcgta atagcgaaga ggcccgcacc 5640gatcgccctt cccaacagtt gcgcagcctg aatggcgaat gggacgcgcc ctgtagcggc 5700gcattaagcg cggcgggtgt ggtggttacg cgcagcgtga ccgctacact tgccagcgcc 5760ctagcgcccg ctcctttcgc tttcttccct tcctttctcg ccacgttcgc cggctttccc 5820cgtcaagctc taaatcgggg gctcccttta gggttccgat ttagtgcttt acggcacctc 5880gaccccaaaa aacttgatta gggtgatggt tcacgtagtg ggccatcgcc ctgatagacg 5940gtttttcgcc ctttgacgtt ggagtccacg ttctttaata gtggactctt gttccaaact 6000ggaacaacac tcaaccctat ctcggtctat tcttttgatt tataagggat tttgccgatt 6060tcggcctatt ggttaaaaaa tgagctgatt taacaaaaat ttaacgcgaa ttttaacaaa 6120atattaacgc ttacaattta ggtggcactt ttcggggaaa tgtgcgcgga acccctattt 6180gtttattttt ctaaatacat tcaaatatgt atccgctcat gagacaataa ccctgataaa 6240tgcttcaata atattgaaaa aggaagagta tgagtattca acatttccgt gtcgccctta 6300ttcccttttt tgcggcattt tgccttcctg tttttgctca cccagaaacg ctggtgaaag 6360taaaagatgc tgaagatcag ttgggtgcac gagtgggtta catcgaactg gatctcaaca 6420gcggtaagat ccttgagagt tttcgccccg aagaacgttt tccaatgatg agcactttta 6480aagttctgct atgtggcgcg gtattatccc gtattgacgc cgggcaagag caactcggtc 6540gccgcataca ctattctcag aatgacttgg ttgagtactc accagtcaca gaaaagcatc 6600ttacggatgg catgacagta agagaattat gcagtgctgc cataaccatg agtgataaca 6660ctgcggccaa cttacttctg acaacgatcg gaggaccgaa ggagctaacc gcttttttgc 6720acaacatggg ggatcatgta actcgccttg atcgttggga accggagctg aatgaagcca 6780taccaaacga cgagcgtgac accacgatgc ctgtagcaat ggcaacaacg ttgcgcaaac 6840tattaactgg cgaactactt actctagctt cccggcaaca attaatagac tggatggagg 6900cggataaagt tgcaggacca cttctgcgct cggcccttcc ggctggctgg tttattgctg 6960ataaatctgg agccggtgag cgtgggtctc gcggtatcat tgcagcactg gggccagatg 7020gtaagccctc ccgtatcgta gttatctaca cgacggggag tcaggcaact atggatgaac 7080gaaatagaca gatcgctgag ataggtgcct cactgattaa gcattggtaa ctgtcagacc 7140aagtttactc atatatactt tagattgatt taaaacttca tttttaattt aaaaggatct 7200aggtgaagat cctttttgat aatctcatga ccaaaatccc ttaacgtgag ttttcgttcc 7260actgagcgtc agaccccgta gaaaagatca aaggatcttc ttgagatcct ttttttctgc 7320gcgtaatctg ctgcttgcaa acaaaaaaac caccgctacc agcggtggtt tgtttgccgg 7380atcaagagct accaactctt tttccgaagg taactggctt cagcagagcg cagataccaa 7440atactgttct tctagtgtag ccgtagttag gccaccactt caagaactct gtagcaccgc 7500ctacatacct cgctctgcta atcctgttac cagtggctgc tgccagtggc gataagtcgt 7560gtcttaccgg gttggactca agacgatagt taccggataa ggcgcagcgg tcgggctgaa 7620cggggggttc gtgcacacag cccagcttgg agcgaacgac ctacaccgaa ctgagatacc 7680tacagcgtga gctatgagaa agcgccacgc ttcccgaagg gagaaaggcg gacaggtatc 7740cggtaagcgg cagggtcgga acaggagagc gcacgaggga gcttccaggg ggaaacgcct 7800ggtatcttta tagtcctgtc gggtttcgcc acctctgact tgagcgtcga tttttgtgat 7860gctcgtcagg ggggcggagc ctatggaaaa acgccagcaa cgcggccttt ttacggttcc 7920tggccttttg ctggcctttt gctcacatgt tctttcctgc gttatcccct gattctgtgg 7980ataaccgtat taccgccttt gagtgagctg ataccgctcg ccgcagccga acgaccgagc 8040gcagcgagtc agtgagcgag gaagcggaag agcgcccaat acgcaaaccg cctctccccg 8100cgcgttggcc gattcattaa tgcagctggc acgacaggtt tcccgactgg aaagcgggca 8160gtgagcgcaa cgcaattaat gtgagttagc tcactcatta ggcaccccag gctttacact 8220ttatgcttcc ggctcgtatg ttgtgtggaa ttgtgagcgg ataacaattt cacacaggaa 8280acagctatga ccatgattac gccaagcgcg caattaaccc tcactaaagg gaacaaaagc 8340tggagctgca agctt 8355 117 6975 DNA Artificial Sequence pLenti6/V5 117aatgtagtct tatgcaatac tcttgtagtc ttgcaacatg gtaacgatga gttagcaaca 60tgccttacaa ggagagaaaa agcaccgtgc atgccgattg gtggaagtaa ggtggtacga 120tcgtgcctta ttaggaaggc aacagacggg tctgacatgg attggacgaa ccactgaatt 180gccgcattgc agagatattg tatttaagtg cctagctcga tacataaacg ggtctctctg 240gttagaccag atctgagcct gggagctctc tggctaacta gggaacccac tgcttaagcc 300tcaataaagc ttgccttgag tgcttcaagt agtgtgtgcc cgtctgttgt gtgactctgg 360taactagaga tccctcagac ccttttagtc agtgtggaaa atctctagca gtggcgcccg 420aacagggact tgaaagcgaa agggaaacca gaggagctct ctcgacgcag gactcggctt 480gctgaagcgc gcacggcaag aggcgagggg cggcgactgg tgagtacgcc aaaaattttg 540actagcggag gctagaagga gagagatggg tgcgagagcg tcagtattaa gcgggggaga 600attagatcgc gatgggaaaa aattcggtta aggccagggg gaaagaaaaa atataaatta 660aaacatatag tatgggcaag cagggagcta gaacgattcg cagttaatcc tggcctgtta 720gaaacatcag aaggctgtag acaaatactg ggacagctac aaccatccct tcagacagga 780tcagaagaac ttagatcatt atataataca gtagcaaccc tctattgtgt gcatcaaagg 840atagagataa aagacaccaa ggaagcttta gacaagatag aggaagagca aaacaaaagt 900aagaccaccg cacagcaagc ggccgctgat cttcagacct ggaggaggag atatgaggga 960caattggaga agtgaattat ataaatataa agtagtaaaa attgaaccat taggagtagc 1020acccaccaag gcaaagagaa gagtggtgca gagagaaaaa agagcagtgg gaataggagc 1080tttgttcctt gggttcttgg gagcagcagg aagcactatg ggcgcagcgt caatgacgct 1140gacggtacag gccagacaat tattgtctgg tatagtgcag cagcagaaca atttgctgag 1200ggctattgag gcgcaacagc atctgttgca actcacagtc tggggcatca agcagctcca 1260ggcaagaatc ctggctgtgg aaagatacct aaaggatcaa cagctcctgg ggatttgggg 1320ttgctctgga aaactcattt gcaccactgc tgtgccttgg aatgctagtt ggagtaataa 1380atctctggaa cagatttgga atcacacgac ctggatggag tgggacagag aaattaacaa 1440ttacacaagc ttaatacact ccttaattga agaatcgcaa aaccagcaag aaaagaatga 1500acaagaatta ttggaattag ataaatgggc aagtttgtgg aattggttta acataacaaa 1560ttggctgtgg tatataaaat tattcataat gatagtagga ggcttggtag gtttaagaat 1620agtttttgct gtactttcta tagtgaatag agttaggcag ggatattcac cattatcgtt 1680tcagacccac ctcccaaccc cgaggggacc cgacaggccc gaaggaatag aagaagaagg 1740tggagagaga gacagagaca gatccattcg attagtgaac ggatctcgac ggtatcgata 1800agcttgggag ttccgcgtta cataacttac ggtaaatggc ccgcctggct gaccgcccaa 1860cgacccccgc ccattgacgt caataatgac gtatgttccc atagtaacgc caatagggac 1920tttccattga cgtcaatggg tggagtattt acggtaaact gcccacttgg cagtacatca 1980agtgtatcat atgccaagta cgccccctat tgacgtcaat gacggtaaat ggcccgcctg 2040gcattatgcc cagtacatga ccttatggga ctttcctact tggcagtaca tctacgtatt 2100agtcatcgct attaccatgg tgatgcggtt ttggcagtac atcaatgggc gtggatagcg 2160gtttgactca cggggatttc caagtctcca ccccattgac gtcaatggga gtttgttttg 2220gcaccaaaat caacgggact ttccaaaatg tcgtaacaac tccgccccat tgacgcaaat 2280gggcggtagg cgtgtacggt gggaggtcta tataagcaga gctcgtttag tgaaccgtca 2340gatcgcctgg agacgccatc cacgctgttt tgacctccat agaagacacc gactctagag 2400gatccactag tccagtgtgg tggaattctg cagatatcca gcacagtggc ggccgctcga 2460gtctagaggg cccgcggttc gaaggtaagc ctatccctaa ccctctcctc ggtctcgatt 2520ctacgcgtac cggttagtaa tgagtttgga attaattctg tggaatgtgt gtcagttagg 2580gtgtggaaag tccccaggct ccccaggcag gcagaagtat gcaaagcatg catctcaatt 2640agtcagcaac caggtgtgga aagtccccag gctccccagc aggcagaagt atgcaaagca 2700tgcatctcaa ttagtcagca accatagtcc cgcccctaac tccgcccatc ccgcccctaa 2760ctccgcccag ttccgcccat tctccgcccc atggctgact aatttttttt atttatgcag 2820aggccgaggc cgcctctgcc tctgagctat tccagaagta gtgaggaggc ttttttggag 2880gcctaggctt ttgcaaaaag ctcccgggag cttgtatatc cattttcgga tctgatcagc 2940acgtgttgac aattaatcat cggcatagta tatcggcata gtataatacg acaaggtgag 3000gaactaaacc atggccaagc ctttgtctca agaagaatcc accctcattg aaagagcaac 3060ggctacaatc aacagcatcc ccatctctga agactacagc gtcgccagcg cagctctctc 3120tagcgacggc cgcatcttca ctggtgtcaa tgtatatcat tttactgggg gaccttgtgc 3180agaactcgtg gtgctgggca ctgctgctgc tgcggcagct ggcaacctga cttgtatcgt 3240cgcgatcgga aatgagaaca ggggcatctt gagcccctgc ggacggtgcc gacaggtgct 3300tctcgatctg catcctggga tcaaagccat agtgaaggac agtgatggac agccgacggc 3360agttgggatt cgtgaattgc tgccctctgg ttatgtgtgg gagggctaag cacaattcga 3420gctcggtacc tttaagacca atgacttaca aggcagctgt agatcttagc cactttttaa 3480aagaaaaggg gggactggaa gggctaattc actcccaacg aagacaagat ctgctttttg 3540cttgtactgg gtctctctgg ttagaccaga tctgagcctg ggagctctct ggctaactag 3600ggaacccact gcttaagcct caataaagct tgccttgagt gcttcaagta gtgtgtgccc 3660gtctgttgtg tgactctggt aactagagat ccctcagacc cttttagtca gtgtggaaaa 3720tctctagcag tagtagttca tgtcatctta ttattcagta tttataactt gcaaagaaat 3780gaatatcaga gagtgagagg aacttgttta ttgcagctta taatggttac aaataaagca 3840atagcatcac aaatttcaca aataaagcat ttttttcact gcattctagt tgtggtttgt 3900ccaaactcat caatgtatct tatcatgtct ggctctagct atcccgcccc taactccgcc 3960catcccgccc ctaactccgc ccagttccgc ccattctccg ccccatggct gactaatttt 4020ttttatttat gcagaggccg aggccgcctc ggcctctgag ctattccaga agtagtgagg 4080aggctttttt ggaggcctag ggacgtaccc aattcgccct atagtgagtc gtattacgcg 4140cgctcactgg ccgtcgtttt acaacgtcgt gactgggaaa accctggcgt tacccaactt 4200aatcgccttg cagcacatcc ccctttcgcc agctggcgta atagcgaaga ggcccgcacc 4260gatcgccctt cccaacagtt gcgcagcctg aatggcgaat gggacgcgcc ctgtagcggc 4320gcattaagcg cggcgggtgt ggtggttacg cgcagcgtga ccgctacact tgccagcgcc 4380ctagcgcccg ctcctttcgc tttcttccct tcctttctcg ccacgttcgc cggctttccc 4440cgtcaagctc taaatcgggg gctcccttta gggttccgat ttagtgcttt acggcacctc 4500gaccccaaaa aacttgatta gggtgatggt tcacgtagtg ggccatcgcc ctgatagacg 4560gtttttcgcc ctttgacgtt ggagtccacg ttctttaata gtggactctt gttccaaact 4620ggaacaacac tcaaccctat ctcggtctat tcttttgatt tataagggat tttgccgatt 4680tcggcctatt ggttaaaaaa tgagctgatt taacaaaaat ttaacgcgaa ttttaacaaa 4740atattaacgc ttacaattta ggtggcactt ttcggggaaa tgtgcgcgga acccctattt 4800gtttattttt ctaaatacat tcaaatatgt atccgctcat gagacaataa ccctgataaa 4860tgcttcaata atattgaaaa aggaagagta tgagtattca acatttccgt gtcgccctta 4920ttcccttttt tgcggcattt tgccttcctg tttttgctca cccagaaacg ctggtgaaag 4980taaaagatgc tgaagatcag ttgggtgcac gagtgggtta catcgaactg gatctcaaca 5040gcggtaagat ccttgagagt tttcgccccg aagaacgttt tccaatgatg agcactttta 5100aagttctgct atgtggcgcg gtattatccc gtattgacgc cgggcaagag caactcggtc 5160gccgcataca ctattctcag aatgacttgg ttgagtactc accagtcaca gaaaagcatc 5220ttacggatgg catgacagta agagaattat gcagtgctgc cataaccatg agtgataaca 5280ctgcggccaa cttacttctg acaacgatcg gaggaccgaa ggagctaacc gcttttttgc 5340acaacatggg ggatcatgta actcgccttg atcgttggga accggagctg aatgaagcca 5400taccaaacga cgagcgtgac accacgatgc ctgtagcaat ggcaacaacg ttgcgcaaac 5460tattaactgg cgaactactt actctagctt cccggcaaca attaatagac tggatggagg 5520cggataaagt tgcaggacca cttctgcgct cggcccttcc ggctggctgg tttattgctg 5580ataaatctgg agccggtgag cgtgggtctc gcggtatcat tgcagcactg gggccagatg 5640gtaagccctc ccgtatcgta gttatctaca cgacggggag tcaggcaact atggatgaac 5700gaaatagaca gatcgctgag ataggtgcct cactgattaa gcattggtaa ctgtcagacc 5760aagtttactc atatatactt tagattgatt taaaacttca tttttaattt aaaaggatct 5820aggtgaagat cctttttgat aatctcatga ccaaaatccc ttaacgtgag ttttcgttcc 5880actgagcgtc agaccccgta gaaaagatca aaggatcttc ttgagatcct ttttttctgc 5940gcgtaatctg ctgcttgcaa acaaaaaaac caccgctacc agcggtggtt tgtttgccgg 6000atcaagagct accaactctt tttccgaagg taactggctt cagcagagcg cagataccaa 6060atactgttct tctagtgtag ccgtagttag gccaccactt caagaactct gtagcaccgc 6120ctacatacct cgctctgcta atcctgttac cagtggctgc tgccagtggc gataagtcgt 6180gtcttaccgg gttggactca agacgatagt taccggataa ggcgcagcgg tcgggctgaa 6240cggggggttc gtgcacacag cccagcttgg agcgaacgac ctacaccgaa ctgagatacc 6300tacagcgtga gctatgagaa agcgccacgc ttcccgaagg gagaaaggcg gacaggtatc 6360cggtaagcgg cagggtcgga acaggagagc gcacgaggga gcttccaggg ggaaacgcct 6420ggtatcttta tagtcctgtc gggtttcgcc acctctgact tgagcgtcga tttttgtgat 6480gctcgtcagg ggggcggagc ctatggaaaa acgccagcaa cgcggccttt ttacggttcc 6540tggccttttg ctggcctttt gctcacatgt tctttcctgc gttatcccct gattctgtgg 6600ataaccgtat taccgccttt gagtgagctg ataccgctcg ccgcagccga acgaccgagc 6660gcagcgagtc agtgagcgag gaagcggaag agcgcccaat acgcaaaccg cctctccccg 6720cgcgttggcc gattcattaa tgcagctggc acgacaggtt tcccgactgg aaagcgggca 6780gtgagcgcaa cgcaattaat gtgagttagc tcactcatta ggcaccccag gctttacact 6840ttatgcttcc ggctcgtatg ttgtgtggaa ttgtgagcgg ataacaattt cacacaggaa 6900acagctatga ccatgattac gccaagcgcg caattaaccc tcactaaagg gaacaaaagc 6960tggagctgca agctt 6975 118 7428 DNA Artificial Sequence pLenti3/V5-TREx118 aatgtagtct tatgcaatac tcttgtagtc ttgcaacatg gtaacgatga gttagcaaca 60tgccttacaa ggagagaaaa agcaccgtgc atgccgattg gtggaagtaa ggtggtacga 120tcgtgcctta ttaggaaggc aacagacggg tctgacatgg attggacgaa ccactgaatt 180gccgcattgc agagatattg tatttaagtg cctagctcga tacataaacg ggtctctctg 240gttagaccag atctgagcct gggagctctc tggctaacta gggaacccac tgcttaagcc 300tcaataaagc ttgccttgag tgcttcaagt agtgtgtgcc cgtctgttgt gtgactctgg 360taactagaga tccctcagac ccttttagtc agtgtggaaa atctctagca gtggcgcccg 420aacagggact tgaaagcgaa agggaaacca gaggagctct ctcgacgcag gactcggctt 480gctgaagcgc gcacggcaag aggcgagggg cggcgactgg tgagtacgcc aaaaattttg 540actagcggag gctagaagga gagagatggg tgcgagagcg tcagtattaa gcgggggaga 600attagatcgc gatgggaaaa aattcggtta aggccagggg gaaagaaaaa atataaatta 660aaacatatag tatgggcaag cagggagcta gaacgattcg cagttaatcc tggcctgtta 720gaaacatcag aaggctgtag acaaatactg ggacagctac aaccatccct tcagacagga 780tcagaagaac ttagatcatt atataataca gtagcaaccc tctattgtgt gcatcaaagg 840atagagataa aagacaccaa ggaagcttta gacaagatag aggaagagca aaacaaaagt 900aagaccaccg cacagcaagc ggccgctgat cttcagacct ggaggaggag atatgaggga 960caattggaga agtgaattat ataaatataa agtagtaaaa attgaaccat taggagtagc 1020acccaccaag gcaaagagaa gagtggtgca gagagaaaaa agagcagtgg gaataggagc 1080tttgttcctt gggttcttgg gagcagcagg aagcactatg ggcgcagcgt caatgacgct 1140gacggtacag gccagacaat tattgtctgg tatagtgcag cagcagaaca atttgctgag 1200ggctattgag gcgcaacagc atctgttgca actcacagtc tggggcatca agcagctcca 1260ggcaagaatc ctggctgtgg aaagatacct aaaggatcaa cagctcctgg ggatttgggg 1320ttgctctgga aaactcattt gcaccactgc tgtgccttgg aatgctagtt ggagtaataa 1380atctctggaa cagatttgga atcacacgac ctggatggag tgggacagag aaattaacaa 1440ttacacaagc ttaatacact ccttaattga agaatcgcaa aaccagcaag aaaagaatga 1500acaagaatta ttggaattag ataaatgggc aagtttgtgg aattggttta acataacaaa 1560ttggctgtgg tatataaaat tattcataat gatagtagga ggcttggtag gtttaagaat 1620agtttttgct gtactttcta tagtgaatag agttaggcag ggatattcac cattatcgtt 1680tcagacccac ctcccaaccc cgaggggacc cgacaggccc gaaggaatag aagaagaagg 1740tggagagaga gacagagaca gatccattcg attagtgaac ggatctcgac ggtatcgata 1800agcttgggag ttccgcgtta cataacttac ggtaaatggc ccgcctggct gaccgcccaa 1860cgacccccgc ccattgacgt caataatgac gtatgttccc atagtaacgc caatagggac 1920tttccattga cgtcaatggg tggagtattt acggtaaact gcccacttgg cagtacatca 1980agtgtatcat atgccaagta cgccccctat tgacgtcaat gacggtaaat ggcccgcctg 2040gcattatgcc cagtacatga ccttatggga ctttcctact tggcagtaca tctacgtatt 2100agtcatcgct attaccatgg tgatgcggtt ttggcagtac atcaatgggc gtggatagcg 2160gtttgactca cggggatttc caagtctcca ccccattgac gtcaatggga gtttgttttg 2220gcaccaaaat caacgggact ttccaaaatg tcgtaacaac tccgccccat tgacgcaaat 2280gggcggtagg cgtgtacggt gggaggtcta tataagcaga gctctcccta tcagtgatag 2340agatctccct atcagtgata gagatcgtcg acgagctcgt ttagtgaacc gtcagatcgc 2400ctggagacgc catccacgct gttttgacct ccatagaaga caccgggacc gatccagcct 2460ccggactcta gaggatccct accggtgata tcctcgagtc tagagggccc gcggttcgaa 2520ggtaagccta tccctaaccc tctcctcggt ctcgattcta cgcgtaccgg ttagtaatga 2580gtttggaatt aattctgtgg aatgtgtgtc agttagggtg tggaaagtcc ccaggctccc 2640caggcaggca gaagtatgca aagcatgcat ctcaattagt cagcaaccag gtgtggaaag 2700tccccaggct ccccagcagg cagaagtatg caaagcatgc atctcaatta gtcagcaacc 2760atagtcccgc ccctaactcc gcccatcccg cccctaactc cgcccagttc cgcccattct 2820ccgccccatg gctgactaat tttttttatt tatgcagagg ccgaggccgc ctctgcctct 2880gagctattcc agaagtagtg aggaggcttt tttggaggcc taggcttttg caaaaagctc 2940cccctgttga caattaatca tcggcatagt atatcggcat agtataatac gacaaggtga 3000ggaactaaac catggcctca attgaacaag atggattgca cgcaggttct ccggccgctt 3060gggtggagag gctattcggc tatgactggg cacaacagac aatcggctgc tctgatgccg 3120ccgtgttccg gctgtcagcg caggggcgcc cggttctttt tgtcaagacc gacctgtccg 3180gtgccctgaa tgaactgcag gacgaggcag cgcggctatc gtggctggcc acgacgggcg 3240ttccttgcgc agctgtgctc gacgttgtca ctgaagcggg aagggactgg ctgctattgg 3300gcgaagtgcc ggggcaggat ctcctgtcat ctcaccttgc tcctgccgag aaagtatcca 3360tcatggctga tgcaatgcgg cggctgcata cgcttgatcc ggctacctgc ccattcgacc 3420accaagcgaa acatcgcatc gagcgagcac gtactcggat ggaagccggt cttgtcgatc 3480aggatgatct ggacgaagag catcaggggc tcgcgccagc cgaactgttc gccaggctca 3540aggcgcgcat gcccgacggc gaggatctcg tcgtgaccca tggcgatgcc tgcttgccga 3600atatcatggt ggaaaatggc cgcttttctg gattcatcga ctgtggccgg ctgggtgtgg 3660cggaccgcta tcaggacata gcgttggcta cccgtgatat tgctgaagag cttggcggcg 3720aatgggctga ccgcttcctc gtgctttacg gtatcgccgc tcccgattcg cagcgcatcg 3780ccttctatcg ccttcttgac gagttcttct gagcgggact ctggggttcg aaatgaccga 3840ccaagcgacg cccaacctgc catcacgagt ttaaactggt acctttaaga ccaatgactt 3900acaaggcagc tgtagatctt agccactttt taaaagaaaa ggggggactg gaagggctaa 3960ttcactccca acgaagacaa gatctgcttt ttgcttgtac tgggtctctc tggttagacc 4020agatctgagc ctgggagctc tctggctaac tagggaaccc actgcttaag cctcaataaa 4080gcttgccttg agtgcttcaa gtagtgtgtg cccgtctgtt gtgtgactct ggtaactaga 4140gatccctcag acccttttag tcagtgtgga aaatctctag cagtagtagt tcatgtcatc 4200ttattattca gtatttataa cttgcaaaga aatgaatatc agagagtgag aggaacttgt 4260ttattgcagc ttataatggt tacaaataaa gcaatagcat cacaaatttc acaaataaag 4320catttttttc actgcattct agttgtggtt tgtccaaact catcaatgta tcttatcatg 4380tctggctcta gctatcccgc ccctaactcc gcccatcccg cccctaactc cgcccagttc 4440cgcccattct ccgccccatg gctgactaat tttttttatt tatgcagagg ccgaggccgc 4500ctcggcctct gagctattcc agaagtagtg aggaggcttt tttggaggcc tagggacgta 4560cccaattcgc cctatagtga gtcgtattac gcgcgctcac tggccgtcgt tttacaacgt 4620cgtgactggg aaaaccctgg cgttacccaa cttaatcgcc ttgcagcaca tccccctttc 4680gccagctggc gtaatagcga agaggcccgc accgatcgcc cttcccaaca gttgcgcagc 4740ctgaatggcg aatgggacgc gccctgtagc ggcgcattaa gcgcggcggg tgtggtggtt 4800acgcgcagcg tgaccgctac acttgccagc gccctagcgc ccgctccttt cgctttcttc 4860ccttcctttc tcgccacgtt cgccggcttt ccccgtcaag ctctaaatcg ggggctccct 4920ttagggttcc gatttagtgc tttacggcac ctcgacccca aaaaacttga ttagggtgat 4980ggttcacgta gtgggccatc gccctgatag acggtttttc gccctttgac gttggagtcc 5040acgttcttta atagtggact cttgttccaa actggaacaa cactcaaccc tatctcggtc 5100tattcttttg atttataagg gattttgccg atttcggcct attggttaaa aaatgagctg 5160atttaacaaa aatttaacgc gaattttaac aaaatattaa cgcttacaat ttaggtggca 5220cttttcgggg aaatgtgcgc ggaaccccta tttgtttatt tttctaaata cattcaaata 5280tgtatccgct catgagacaa taaccctgat aaatgcttca ataatattga aaaaggaaga 5340gtatgagtat tcaacatttc cgtgtcgccc ttattccctt ttttgcggca ttttgccttc 5400ctgtttttgc tcacccagaa acgctggtga aagtaaaaga tgctgaagat cagttgggtg 5460cacgagtggg ttacatcgaa ctggatctca acagcggtaa gatccttgag agttttcgcc 5520ccgaagaacg ttttccaatg atgagcactt ttaaagttct gctatgtggc gcggtattat 5580cccgtattga cgccgggcaa gagcaactcg gtcgccgcat acactattct cagaatgact 5640tggttgagta ctcaccagtc acagaaaagc atcttacgga tggcatgaca gtaagagaat 5700tatgcagtgc tgccataacc atgagtgata acactgcggc caacttactt ctgacaacga 5760tcggaggacc gaaggagcta accgcttttt tgcacaacat gggggatcat gtaactcgcc 5820ttgatcgttg ggaaccggag ctgaatgaag ccataccaaa cgacgagcgt gacaccacga 5880tgcctgtagc aatggcaaca acgttgcgca aactattaac tggcgaacta cttactctag 5940cttcccggca acaattaata gactggatgg aggcggataa agttgcagga ccacttctgc 6000gctcggccct tccggctggc tggtttattg ctgataaatc tggagccggt gagcgtgggt 6060ctcgcggtat cattgcagca ctggggccag atggtaagcc ctcccgtatc gtagttatct 6120acacgacggg gagtcaggca actatggatg aacgaaatag acagatcgct gagataggtg 6180cctcactgat taagcattgg taactgtcag accaagttta ctcatatata ctttagattg 6240atttaaaact tcatttttaa tttaaaagga tctaggtgaa gatccttttt gataatctca 6300tgaccaaaat cccttaacgt gagttttcgt tccactgagc gtcagacccc gtagaaaaga 6360tcaaaggatc ttcttgagat cctttttttc tgcgcgtaat ctgctgcttg caaacaaaaa 6420aaccaccgct accagcggtg gtttgtttgc cggatcaaga gctaccaact ctttttccga 6480aggtaactgg cttcagcaga gcgcagatac caaatactgt tcttctagtg tagccgtagt 6540taggccacca cttcaagaac tctgtagcac cgcctacata cctcgctctg ctaatcctgt 6600taccagtggc tgctgccagt ggcgataagt cgtgtcttac cgggttggac tcaagacgat 6660agttaccgga taaggcgcag cggtcgggct gaacgggggg ttcgtgcaca cagcccagct 6720tggagcgaac gacctacacc gaactgagat acctacagcg tgagctatga gaaagcgcca 6780cgcttcccga agggagaaag gcggacaggt atccggtaag cggcagggtc ggaacaggag 6840agcgcacgag ggagcttcca gggggaaacg cctggtatct ttatagtcct gtcgggtttc 6900gccacctctg acttgagcgt cgatttttgt gatgctcgtc aggggggcgg agcctatgga 6960aaaacgccag caacgcggcc tttttacggt tcctggcctt ttgctggcct tttgctcaca 7020tgttctttcc tgcgttatcc cctgattctg tggataaccg tattaccgcc tttgagtgag 7080ctgataccgc tcgccgcagc cgaacgaccg agcgcagcga gtcagtgagc gaggaagcgg 7140aagagcgccc aatacgcaaa ccgcctctcc ccgcgcgttg gccgattcat taatgcagct 7200ggcacgacag gtttcccgac tggaaagcgg gcagtgagcg caacgcaatt aatgtgagtt 7260agctcactca ttaggcaccc caggctttac actttatgct tccggctcgt atgttgtgtg 7320gaattgtgag cggataacaa tttcacacag gaaacagcta tgaccatgat tacgccaagc 7380gcgcaattaa ccctcactaa agggaacaaa agctggagct gcaagctt 7428 119 1474 DNAUnknown Nucleic acid fragment containing the tetracycline repressorcoding sequence 119 agcttggtac ccggggatcc tctagggcct ctgagctattccagaagtag tgaagaggct 60 tttttggagg cctaggcttt tgcaaaaagc tccggatcgatcctgagaac ttcagggtga 120 gtttggggac ccttgattgt tctttctttt tcgctattgtaaaattcatg ttatatggag 180 ggggcaaagt tttcagggtg ttgtttagaa tgggaagatgtcccttgtat caccatggac 240 cctcatgata attttgtttc tttcactttc tactctgttgacaaccattg tctcctctta 300 ttttcttttc attttctgta actttttcgt taaactttagcttgcatttg taacgaattt 360 ttaaattcac ttttgtttat ttgtcagatt gtaagtactttctctaatca cttttttttc 420 aaggcaatca gggtatatta tattgtactt cagcacagttttagagaaca attgttataa 480 ttaaatgata aggtagaata tttctgcata taaattctggctggcgtgga aatattctta 540 ttggtagaaa caactacatc ctggtcatca tcctgcctttctctttatgg ttacaatgat 600 atacactgtt tgagatgagg ataaaatact ctgagtccaaaccgggcccc tctgctaacc 660 atgttcatgc cttcttcttt ttcctacagc tcctgggcaacgtgctggtt attgtgctgt 720 ctcatcattt tggcaaagaa ttgtaatacg actcactatagggcgaattg atatgtctag 780 attagataaa agtaaagtga ttaacagcgc attagagctgcttaatgagg tcggaatcga 840 aggtttaaca acccgtaaac tcgcccagaa gctaggtgtagagcagccta cattgtattg 900 gcatgtaaaa aataagcggg ctttgctcga cgccttagccattgagatgt tagataggca 960 ccatactcac ttttgccctt tagaagggga aagctggcaagattttttac gtaataacgc 1020 taaaagtttt agatgtgctt tactaagtca tcgcgatggagcaaaagtac atttaggtac 1080 acggcctaca gaaaaacagt atgaaactct cgaaaatcaattagcctttt tatgccaaca 1140 aggtttttca ctagagaatg cattatatgc actcagcgctgtggggcatt ttactttagg 1200 ttgcgtattg gaagatcaag agcatcaagt cgctaaagaagaaagggaaa cacctactac 1260 tgatagtatg ccgccattat tacgacaagc tatcgaattatttgatcacc aaggtgcaga 1320 gccagccttc ttattcggcc ttgaattgat catatgcggattagaaaaac aacttaaatg 1380 tgaaagtggg tccgcgtaca gcggatcccg ggaattctagagggcccgcg gttcgaacaa 1440 aaactcatct cagaagagga tctgaatatg cata 1474120 7056 DNA Artificial Sequence pRRL6/V5 also referred to as pLenti6/V5120 aatgtagtct tatgcaatac tcttgtagtc ttgcaacatg gtaacgatga gttagcaaca 60tgccttacaa ggagagaaaa agcaccgtgc atgccgattg gtggaagtaa ggtggtacga 120tcgtgcctta ttaggaaggc aacagacggg tctgacatgg attggacgaa ccactgaatt 180gccgcattgc agagatattg tatttaagtg cctagctcga tacaataaac gggtctctct 240ggttagacca gatctgagcc tgggagctct ctggctaact agggaaccca ctgcttaagc 300ctcaataaag cttgccttga gtgcttcaag tagtgtgtgc ccgtctgttg tgtgactctg 360gtaactagag atccctcaga cccttttagt cagtgtggaa aatctctagc agtggcgccc 420gaacagggac ctgaaagcga aagggaaacc agagctctct cgacgcagga ctcggcttgc 480tgaagcgcgc acggcaagag gcgaggggcg gcgactggtg agtacgccaa aaattttgac 540tagcggaggc tagaaggaga gagatgggtg cgagagcgtc agtattaagc gggggagaat 600tagatcgcga tgggaaaaaa ttcggttaag gccaggggga aagaaaaaat ataaattaaa 660acatatagta tgggcaagca gggagctaga acgattcgca gttaatcctg gcctgttaga 720aacatcagaa ggctgtagac aaatactggg acagctacaa ccatcccttc agacaggatc 780agaagaactt agatcattat ataatacagt agcaaccctc tattgtgtgc atcaaaggat 840agagataaaa gacaccaagg aagctttaga caagatagag gaagagcaaa acaaaagtaa 900gaccaccgca cagcaagcgg ccgctgatct tcagacctgg aggaggagat atgagggaca 960attggagaag tgaattatat aaatataaag tagtaaaaat tgaaccatta ggagtagcac 1020ccaccaaggc aaagagaaga gtggtgcaga gagaaaaaag agcagtggga ataggagctt 1080tgttccttgg gttcttggga gcagcaggaa gcactatggg cgcagcctca atgacgctga 1140cggtacaggc cagacaatta ttgtctggta tagtgcagca gcagaacaat ttgctgaggg 1200ctattgaggc gcaacagcat ctgttgcaac tcacagtctg gggcatcaag cagctccagg 1260caagaatcct ggctgtggaa agatacctaa aggatcaaca gctcctgggg atttggggtt 1320gctctggaaa actcatttgc accactgctg tgccttggaa tgctagttgg agtaataaat 1380ctctggaaca gattggaatc acacgacctg gatggagtgg gacagagaaa ttaacaatta 1440cacaagctta atacactcct taattgaaga atcgcaaaac cagcaagaaa agaatgaaca 1500agaattattg gaattagata aatgggcaag tttgtggaat tggtttaaca taacaaattg 1560gctgtggtat ataaaattat tcataatgat agtaggaggc ttggtaggtt taagaatagt 1620ttttgctgta ctttctatag tgaatagagt taggcaggga tattcaccat tatcgtttca 1680gacccacctc ccaaccccga ggggacccga caggcccgaa ggaatagaag aagaaggtgg 1740agagagagac agagacagat ccattcgatt agtgaacgga tctcgacggt atcgataagc 1800ttgggagttc cgcgttacat aacttacggt aaatggcccg cctggctgac cgcccaacga 1860cccccgccca ttgacgtcaa taatgacgta tgttcccata gtaacgccaa tagggacttt 1920ccattgacgt caatgggtgg agtatttacg gtaaactgcc cacttggcag tacatcaagt 1980gtatcatatg ccaagtacgc cccctattga cgtcaatgac ggtaaatggc ccgcctggca 2040ttatgcccag tacatgacct tatgggactt tcctacttgg cagtacatct acgtattagt 2100catcgctatt accatggtga tgcggttttg gcagtacatc aatgggcgtg gatagcggtt 2160tgactcacgg ggatttccaa gtctccaccc cattgacgtc aatgggagtt tgttttggca 2220ccaaaatcaa cgggactttc caaaatgtcg taacaactcc gccccattga cgcaaatggg 2280cggtaggcgt gtacggtggg aggtctatat aagcagagct cgtttagtga accgtcagat 2340cgcctggaga cgccatccac gctgttttga cctccataga agacaccgac tctagaggat 2400ccactagtcc agtgtggtgg aattctgcag atatccagca cagtggcggc cgctcgagtc 2460tagagggccc gcggttcgaa ggtaagccta tccctaaccc tctcctcggt ctcgattcta 2520cgcgtaccgg ttagtaatga gtttggcctg ctgccggctc tgcggcctct tccgcgtctt 2580cgccttcgcc ctcagacgag tcggatctcc ctttgggccg cctccccgcc tggaattaat 2640tctgtggaat gtgtgtcagt tagggtgtgg aaagtcccca ggctccccag gcaggcagaa 2700gtatgcaaag catgcatctc aattagtcag caaccaggtg tggaaagtcc ccaggctccc 2760cagcaggcag aagtatgcaa agcatgcatc tcaattagtc agcaaccata gtcccgcccc 2820taactccgcc catcccgccc ctaactccgc ccagttccgc ccattctccg ccccatggct 2880gactaatttt ttttatttat gcagaggccg aggccgcctc tgcctctgag ctattccaga 2940agtagtgagg aggctttttt ggaggcctag gcttttgcaa aaagctcccg ggagcttgta 3000tatccatttt cggatctgat cagcacgtgt tgacaattaa tcatcggcat agtatatcgg 3060catagtataa tacgacaagg tgaggaacta aaccatggcc aagcctttgt ctcaagaaga 3120atccaccctc attgaaagag caacggctac aatcaacagc atccccatct ctgaagacta 3180cagcgtcgcc agcgcagctc tctctagcga cggccgcatc ttcactggtg tcaatgtata 3240tcattttact gggggacctt gtgcagaact cgtggtgctg ggcactgctg ctgctgcggc 3300agctggcaac ctgacttgta tcgtcgcgat cggaaatgag aacaggggca tcttgagccc 3360ctgcggacgg tgccgacagg tgcttctcga tctgcatcct gggatcaaag ccatagtgaa 3420ggacagtgat ggacagccga cggcagttgg gattcgtgaa ttgctgccct ctggttatgt 3480gtgggagggc taagcacaat tcgagctcgg tacctttaag accaatgact tacaaggcag 3540ctgtagatct tagccacttt ttaaaagaaa aggggggact ggaagggcta attcactccc 3600aacgaagaca agatctgctt tttgcttgta ctgggtctct ctggttagac cagatctgag 3660cctgggagct ctctggctaa ctagggaacc cactgcttaa gcctcaataa agcttgcctt 3720gagtgcttca agtagtgtgt gcccgtctgt tgtgtgactc tggtaactag agatccctca 3780gaccctttta gtcagtgtgg aaaatctcta gcagtagtag ttcatgtcat cttattattc 3840agtatttata acttgcaaag aaatgaatat cagagagtga gaggaacttg tttattgcag 3900cttataatgg ttacaaataa agcaatagca tcacaaattt cacaaataaa gcattttttt 3960cactgcattc tagttgtggt ttgtccaaac tcatcaatgt atcttatcat gtctggctct 4020agctatcccg cccctaactc cgcccagttc cgcccattct ccgccccatg gctgactaat 4080tttttttatt tatgcagagg ccgaggccgc ctcggcctct gagctattcc agaagtagtg 4140aggaggcttt tttggaggcc taggcttttg cgtcgagacg tacccaattc gccctatagt 4200gagtcgtatt acgcgcgctc actggccgtc gttttacaac gtcgtgactg ggaaaaccct 4260ggcgttaccc aacttaatcg ccttgcagca catccccctt tcgccagctg gcgtaatagc 4320gaagaggccc gcaccgatcg cccttcccaa cagttgcgca gcctgaatgg cgaatggcgc 4380gacgcgccct gtagcggcgc attaagcgcg gcgggtgtgg tggttacgcg cagcgtgacc 4440gctacacttg ccagcgccct agcgcccgct cctttcgctt tcttcccttc ctttctcgcc 4500acgttcgccg gctttccccg tcaagctcta aatcgggggc tccctttagg gttccgattt 4560agtgctttac ggcacctcga ccccaaaaaa cttgattagg gtgatggttc acgtagtggg 4620ccatcgccct gatagacggt ttttcgccct ttgacgttgg agtccacgtt ctttaatagt 4680ggactcttgt tccaaactgg aacaacactc aaccctatct cggtctattc ttttgattta 4740taagggattt tgccgatttc ggcctattgg ttaaaaaatg agctgattta acaaaaattt 4800aacgcgaatt ttaacaaaat attaacgttt acaatttccc aggtggcact tttcggggaa 4860atgtgcgcgg aacccctatt tgtttatttt tctaaataca ttcaaatatg tatccgctca 4920tgagacaata accctgataa atgcttcaat aatattgaaa aaggaagagt atgagtattc 4980aacatttccg tgtcgccctt attccctttt ttgcggcatt ttgccttcct gtttttgctc 5040acccagaaac gctggtgaaa gtaaaagatg ctgaagatca gttgggtgca cgagtgggtt 5100acatcgaact ggatctcaac agcggtaaga tccttgagag ttttcgcccc gaagaacgtt 5160ttccaatgat gagcactttt aaagttctgc tatgtggcgc ggtattatcc cgtattgacg 5220ccgggcaaga gcaactcggt cgccgcatac actattctca gaatgacttg gttgagtact 5280caccagtcac agaaaagcat cttacggatg gcatgacagt aagagaatta tgcagtgctg 5340ccataaccat gagtgataac actgcggcca acttacttct gacaacgatc ggaggaccga 5400aggagctaac cgcttttttg cacaacatgg gggatcatgt aactcgcctt gatcgttggg 5460aaccggagct gaatgaagcc ataccaaacg acgagcgtga caccacgatg cctgtagcaa 5520tggcaacaac gttgcgcaaa ctattaactg gcgaactact tactctagct tcccggcaac 5580aattaataga ctggatggag gcggataaag ttgcaggacc acttctgcgc tcggcccttc 5640cggctggctg gtttattgct gataaatctg gagccggtga gcgtgggtct cgcggtatca 5700ttgcagcact ggggccagat ggtaagccct cccgtatcgt agttatctac acgacgggga 5760gtcaggcaac tatggatgaa cgaaatagac agatcgctga gataggtgcc tcactgatta 5820agcattggta actgtcagac caagtttact catatatact ttagattgat ttaaaacttc 5880atttttaatt taaaaggatc taggtgaaga tcctttttga taatctcatg accaaaatcc 5940cttaacgtga gttttcgttc cactgagcgt cagaccccgt agaaaagatc aaaggatctt 6000cttgagatcc tttttttctg cgcgtaatct gctgcttgca aacaaaaaaa ccaccgctac 6060cagcggtggt ttgtttgccg gatcaagagc taccaactct ttttccgaag gtaactggct 6120tcagcagagc gcagatacca aatactgtcc ttctagtgta gccgtagtta ggccaccact 6180tcaagaactc tgtagcaccg cctacatacc tcgctctgct aatcctgtta ccagtggctg 6240ctgccagtgg cgataagtcg tgtcttaccg ggttggactc aagacgatag ttaccggata 6300aggcgcagcg gtcgggctga acggggggtt cgtgcacaca gcccagcttg gagcgaacga 6360cctacaccga actgagatac ctacagcgtg agctatgaga aagcgccacg cttcccgaag 6420ggagaaaggc ggacaggtat ccggtaagcg gcagggtcgg aacaggagag cgcacgaggg 6480agcttccagg gggaaacgcc tggtatcttt atagtcctgt cgggtttcgc cacctctgac 6540ttgagcgtcg atttttgtga tgctcgtcag gggggcggag cctatggaaa aacgccagca 6600acgcggcctt tttacggttc ctggcctttt gctggccttt tgctcacatg ttctttcctg 6660cgttatcccc tgattctgtg gataaccgta ttaccgcctt tgagtgagct gataccgctc 6720gccgcagccg aacgaccgag cgcagcgagt cagtgagcga ggaagcggaa gagcgcccaa 6780tacgcaaacc gcctctcccc gcgcgttggc cgattcatta atgcagctgg cacgacaggt 6840ttcccgactg gaaagcgggc agtgagcgca acgcaattaa tgtgagttag ctcactcatt 6900aggcacccca ggctttacac tttatgcttc cggctcgtat gttgtgtgga attgtgagcg 6960gataacaatt tcacacagga aacagctatg accatgatta cgccaagcgc gcaattaacc 7020ctcactaaag ggaacaaaag ctggagctgc aagctt 7056 121 172 DNA ArtificialSequence Recombination region of pAd/CMV/V5-DEST 121 ttgacgcaaatgggcggtag gcgtgtacgg tgggaggtct atataagcag agctctctgg 60 ctaactagagaacccactgc ttactggctt atcgaaatta atacgactca ctatagggag 120 acccaagctggctagttaag ctatcaacaa gtttgtacaa aaaagcaggc tn 172 122 135 DNAArtificial Sequence Recombination region of pAd/CMV/V5-DEST 122 nac ccagct ttc ttg tac aaa gtg gtt gat cta gag ggc ccg cgg ttc 48 Pro Ala PheLeu Tyr Lys Val Val Asp Leu Glu Gly Pro Arg Phe 1 5 10 15 gaa ggt aagcct atc cct aac cct ctc ctc ggt ctc gat tct acg cgt 96 Glu Gly Lys ProIle Pro Asn Pro Leu Leu Gly Leu Asp Ser Thr Arg 20 25 30 acc ggt tagtaatgagttt aaacggggga ggctaactga 135 Thr Gly 123 33 PRT ArtificialSequence Recombination region of pAd/CMV/V5-DEST 123 Pro Ala Phe Leu TyrLys Val Val Asp Leu Glu Gly Pro Arg Phe Glu 1 5 10 15 Gly Lys Pro IlePro Asn Pro Leu Leu Gly Leu Asp Ser Thr Arg Thr 20 25 30 Gly 124 197 DNAArtificial Sequence Recombination region of pAd/PL-DEST 124 tatttgtctagggccgcggg gactttgacc gtttacgtgg agactcgccc aggtgttttt 60 ctcaggtgttttccgcgttc cgggtcaaag ttggcgtttt attattatag tcagtcgaag 120 cttggatccggtacctctag aattctcgag cggccgctag cgacatcgat cacaagtttg 180 tacaaaaaagcaggctn 197 125 90 DNA Artificial Sequence Recombination region ofpAd/PL-DEST 125 nacccagctt tcttgtacaa agtggtgatc gattcgacag atcactgaaatgtgtgggcg 60 tggcttaagg gtgggaaaga atatataagg 90 126 560 DNA UnknownOpIE2 promoter sequence 126 ggatcatgat gataaacaat gtatggtgct aatgttgcttcaacaacaat tctgttgaac 60 tgtgttttca tgtttgccaa caagcacctt tatactcggtggcctcccca ccaccaactt 120 ttttgcactg caaaaaaaca cgcttttgca cgcgggcccatacatagtac aaactctacg 180 tttcgtagac tattttacat aaatagtcta caccgttgtatacgctccaa atacactacc 240 acacattgaa cctttttgca gtgcaaaaaa gtacgtgtcggcagtcacgt aggccggcct 300 tatcgggtcg cgtcctgtca cgtacgaatc acattatcggaccggacgag tgttgtctta 360 tcgtgacagg acgccagctt cctgtgttgc taaccgcagccggacgcaac tccttatcgg 420 aacaggacgc gcctccatat cagccgcgcg ttatctcatgcgcgtgaccg gacacgaggc 480 gcccgtcccg cttatcgcgc ctataaatac agcccgcaacgatctggtaa acacagttga 540 acagcatctg ttcgaattta 560 127 147 DNAArtificial Sequence Recombination region of pIB/V5-His-DEST 127cttatcgcgc ctataaatac agcccgcaac gatctggtaa acacagttga acagcatctg 60ttcgaattta aagcttgata tcgaattcct gcagcccagc gctggatcct cgatcacaag 120tttgtacaaa aaagcaggct nnnnnnn 147 128 184 DNA Artificial SequenceRecombination region of pIB/V5-His-DEST 128 nac cca cca gct ttc ttg tacaaa gtg gtg atc gac ccg ggt cta gag 48 Pro Pro Ala Phe Leu Tyr Lys ValVal Ile Asp Pro Gly Leu Glu 1 5 10 15 ggc ccg cgg ttc gaa ggt aag cctatc cct aac cct ctc ctc ggt ctc 96 Gly Pro Arg Phe Glu Gly Lys Pro IlePro Asn Pro Leu Leu Gly Leu 20 25 30 gat tct acg cgt acc ggt cat cat caccat cac cat tga gtttatctga 145 Asp Ser Thr Arg Thr Gly His His His HisHis His 35 40 ctaaatctta gttgtattgt catgttttaa tacaatatg 184 129 43 PRTArtificial Sequence Recombination region of pIB/V5-His-DEST 129 Pro ProAla Phe Leu Tyr Lys Val Val Ile Asp Pro Gly Leu Glu Gly 1 5 10 15 ProArg Phe Glu Gly Lys Pro Ile Pro Asn Pro Leu Leu Gly Leu Asp 20 25 30 SerThr Arg Thr Gly His His His His His His 35 40 130 215 DNA ArtificialSequence Recombination region of pLenti6/V5-DEST 130 tcgtaacaactccgccccat tgacgcaaat gggcggtagg cgtgtacggt gggaggtcta 60 tataagcagagctcgtttag tgaaccgtca gatcgcctgg agacgccatc cacgctgttt 120 tgacctccatagaagacacc gactctagag gatccactag tccagtgtgg tggaattctg 180 cagatatcaacaagtttgta caaaaaagca ggctn 215 131 142 DNA Artificial SequenceRecombination region of pLenti6/V5-DEST 131 nac cca gct ttc ttg tac aaagtg gtt gat atc cag cac agt ggc ggc 48 Pro Ala Phe Leu Tyr Lys Val ValAsp Ile Gln His Ser Gly Gly 1 5 10 15 cgc tcg agt cta gag ggc ccg cggttc gaa ggt aag cct atc cct aac 96 Arg Ser Ser Leu Glu Gly Pro Arg PheGlu Gly Lys Pro Ile Pro Asn 20 25 30 cct ctc ctc ggt ctc gat tct acg cgtacc ggt tag taatgagttt 142 Pro Leu Leu Gly Leu Asp Ser Thr Arg Thr Gly35 40 132 42 PRT Artificial Sequence Recombination region ofpLenti6/V5-DEST 132 Pro Ala Phe Leu Tyr Lys Val Val Asp Ile Gln His SerGly Gly Arg 1 5 10 15 Ser Ser Leu Glu Gly Pro Arg Phe Glu Gly Lys ProIle Pro Asn Pro 20 25 30 Leu Leu Gly Leu Asp Ser Thr Arg Thr Gly 35 40133 217 DNA Artificial Sequence Recombination region of the expressionclone resulting from pLenti6/UbC/V5-DEST x entry clone 133 ttggcgagtgtgttttgtga agttttttag gcaccttttg aaatgtaatc atttgggtca 60 atatgtaattttcagtgtta gactagtaaa ttgtccgcta aattctggcc gtttttggct 120 tttttgttagacgaagcttg gtaccgagct cggatccact agtccagtgt ggtggaattc 180 tgcagatatcaacaagtttg tacaaaaaag caggctn 217 134 142 DNA Artificial SequenceRecombination region of the expression clone resulting frompLenti6/UbC/V5-DEST x entry clone 134 nac cca gct ttc ttg tac aaa gtggtt gat atc cag cac agt ggc ggc 48 Pro Ala Phe Leu Tyr Lys Val Val AspIle Gln His Ser Gly Gly 1 5 10 15 cgc tcg agt cta gag ggc ccg cgg ttcgaa ggt aag cct atc cct aac 96 Arg Ser Ser Leu Glu Gly Pro Arg Phe GluGly Lys Pro Ile Pro Asn 20 25 30 cct ctc ctc ggt ctc gat tct acg cgt accggt tag taatgagttt 142 Pro Leu Leu Gly Leu Asp Ser Thr Arg Thr Gly 35 40135 42 PRT Artificial Sequence Recombination region of the expressionclone resulting from pLenti6/UbC/V5-DEST x entry clone 135 Pro Ala PheLeu Tyr Lys Val Val Asp Ile Gln His Ser Gly Gly Arg 1 5 10 15 Ser SerLeu Glu Gly Pro Arg Phe Glu Gly Lys Pro Ile Pro Asn Pro 20 25 30 Leu LeuGly Leu Asp Ser Thr Arg Thr Gly 35 40 136 1226 DNA Unknown Sequence ofthe UbC promoter 136 cggatctggc ctccgcgccg ggttttggcg cctcccgcgggcgcccccct cctcacggcg 60 agcgctgcca cgtcagacga agggcgcagg agcgtcctgatccttccgcc cggacgctca 120 ggacagcggc ccgctgctca taagactcgg ccttagaaccccagtatcag cagaaggaca 180 ttttaggacg ggacttgggt gactctaggg cactggttttctttccagag agcggaacag 240 gcgaggaaaa gtagtccctt ctcggcgatt ctgcggagggatctccgtgg ggcggtgaac 300 gccgatgatt atataaggac gcgccgggtg tggcacagctagttccgtcg cagccgggat 360 ttgggtcgcg gttcttgttt gtggatcgct gtgatcgtcacttggtgagt agcgggctgc 420 tgggctggcc ggggctttcg tggccgccgg gccgctcggtgggacggaag cgtgtggaga 480 gaccgccaag ggctgtagtc tgggtccgcg agcaaggttgccctgaactg ggggttgggg 540 ggagcgcagc aaaatggcgg ctgttcccga gtcttgaatggaagacgctt gtgaggcggg 600 ctgtgaggtc gttgaaacaa ggtggggggc atggtgggcggcaagaaccc aaggtcttga 660 ggccttcgct aatgcgggaa agctcttatt cgggtgagatgggctggggc accatctggg 720 gaccctgacg tgaagtttgt cactgactgg agaactcggtttgtcgtctg ttgcgggggc 780 ggcagttatg cggtgccgtt gggcagtgca cccgtacctttgggagcgcg cgccctcgtc 840 gtgtcgtgac gtcacccgtt ctgttggctt ataatgcagggtggggccac ctgccggtag 900 gtgtgcggta ggcttttctc cgtcgcagga cgcagggttcgggcctaggg taggctctcc 960 tgaatcgaca ggcgccggac ctctggtgag gggagggataagtgaggcgt cagtttcttt 1020 ggtcggtttt atgtacctat cttcttaagt agctgaagctccggttttga actatgcgct 1080 cggggttggc gagtgtgttt tgtgaagttt tttaggcaccttttgaaatg taatcatttg 1140 ggtcaatatg taattttcag tgttagacta gtaaattgtccgctaaattc tggccgtttt 1200 tggctttttt gttagacgaa gcttgg 1226 137 32 DNAUnknown Directional cloning product of Figure 47 137 cccttcaccatgnnnnnnnn nnnnnnnaag gg 32 138 192 DNA Artificial Sequence Cloningregion of pLenti6/V5-D-Topo 138 tcgtaacaac tccgccccat tgacgcaaatgggcggtagg cgtgtacggt gggaggtcta 60 tataagcaga gctcgtttag tgaaccgtcagatcgcctgg agacgccatc cacgctgttt 120 tgacctccat agaagacacc gactctagaggatccactag tccagtgtgg tggaattgat 180 cccttcacca tg 192 139 101 DNAArtificial Sequence Cloning region of pLenti6/V5-D-Topo 139 aag ggc tcgagt cta gag ggc ccg cgg ttc gaa ggt aag cct atc cct 48 Lys Gly Ser SerLeu Glu Gly Pro Arg Phe Glu Gly Lys Pro Ile Pro 1 5 10 15 aac cct ctcctc ggt ctc gat tct acg cgt acc ggt tag taatgagttt 97 Asn Pro Leu LeuGly Leu Asp Ser Thr Arg Thr Gly 20 25 ggaa 101 140 28 PRT ArtificialSequence Cloning region of pLenti6/V5-D-Topo 140 Lys Gly Ser Ser Leu GluGly Pro Arg Phe Glu Gly Lys Pro Ile Pro 1 5 10 15 Asn Pro Leu Leu GlyLeu Asp Ser Thr Arg Thr Gly 20 25 141 166 DNA Artificial SequenceRecombination region of pcDNA6.2/V5-DEST 141 caaatgggcg gtaggcgtgtacggtgggag gtctatataa gcagagctct ctggctaact 60 agagaaccca ctgcttactggcttatcgaa attaatacga ctcactatag ggagacccaa 120 gctggctagt taagctatcaacaagtttgt acaaaaaagc aggctn 166 142 144 DNA Artificial SequenceRecombination region of pcDNA6.2/V5-DEST 142 tagnac cca gct ttc ttg tacaaa gtg gtt gat cta gag ggc ccg cgg 48 Pro Ala Phe Leu Tyr Lys Val ValAsp Leu Glu Gly Pro Arg 1 5 10 ttc gaa ggt aag cct atc cct aac cct ctcctc ggt ctc gat tct acg 96 Phe Glu Gly Lys Pro Ile Pro Asn Pro Leu LeuGly Leu Asp Ser Thr 15 20 25 30 cgt acc ggt tag taatgagttt aaacgggggaggctaactga aacacg 144 Arg Thr Gly 143 33 PRT Artificial SequenceRecombination region of pcDNA6.2/V5-DEST 143 Pro Ala Phe Leu Tyr Lys ValVal Asp Leu Glu Gly Pro Arg Phe Glu 1 5 10 15 Gly Lys Pro Ile Pro AsnPro Leu Leu Gly Leu Asp Ser Thr Arg Thr 20 25 30 Gly 144 166 DNAArtificial Sequence Recombination region of pcDNA6.2/GFP-DEST 144caaatgggcg gtaggcgtgt acggtgggag gtctatataa gcagagctct ctggctaact 60agagaaccca ctgcttactg gcttatcgaa attaatacga ctcactatag ggagacccaa 120gctggctagt taagctatca acaagtttgt acaaaaaagc aggctn 166 145 213 DNAArtificial Sequence Recombination region of pcDNA6.2/GFP-DEST 145 tagnaccca gct ttc ttg tac aaa gtg gtt gat cta gag ggc ccc gcg 48 Pro Ala PheLeu Tyr Lys Val Val Asp Leu Glu Gly Pro Ala 1 5 10 gct agc aaa gga gaagaa ctt ttc act gga ggt gtc cca att ctt gtt 96 Ala Ser Lys Gly Glu GluLeu Phe Thr Gly Gly Val Pro Ile Leu Val 15 20 25 30 gaa tta gat ggt gatgtt aat ggg cac aaa ttt tct gtc agt gga gag 144 Glu Leu Asp Gly Asp ValAsn Gly His Lys Phe Ser Val Ser Gly Glu 35 40 45 ggt gaa ggt gat gct acatac gga aag ctt acc ctt aaa ttt att tgc 192 Gly Glu Gly Asp Ala Thr TyrGly Lys Leu Thr Leu Lys Phe Ile Cys 50 55 60 act act gga aaa cta cct gtt213 Thr Thr Gly Lys Leu Pro Val 65 146 69 PRT Artificial SequenceRecombination region of pcDNA6.2/GFP-DEST 146 Pro Ala Phe Leu Tyr LysVal Val Asp Leu Glu Gly Pro Ala Ala Ser 1 5 10 15 Lys Gly Glu Glu LeuPhe Thr Gly Gly Val Pro Ile Leu Val Glu Leu 20 25 30 Asp Gly Asp Val AsnGly His Lys Phe Ser Val Ser Gly Glu Gly Glu 35 40 45 Gly Asp Ala Thr TyrGly Lys Leu Thr Leu Lys Phe Ile Cys Thr Thr 50 55 60 Gly Lys Leu Pro Val65 147 307 DNA Artificial Recombination region of pAd/CMV/V5 DEST 147ttgacgcaaa tgggcggtag gcgtgtacgg tgggaggtct atataagcag agctctctgg 60ctaactagag aacccactgc ttactggctt atcgaaatta atacgactca ctatagggag 120acccaagctg gctagttaag ctatcaacaa gtttgtacaa aaaagcaggc tnnac cca 178 Pro1 gct ttc ttg tac aaa gtg gtt gat cta gag ggc ccg cgg ttc gaa ggt 226Ala Phe Leu Tyr Lys Val Val Asp Leu Glu Gly Pro Arg Phe Glu Gly 5 10 15aag cct atc cct aac cct ctc ctc ggt ctc gat tct acg cgt acc ggt 274 LysPro Ile Pro Asn Pro Leu Leu Gly Leu Asp Ser Thr Arg Thr Gly 20 25 30 tagtaatgagttt aaacggggga ggctaactga 307 148 287 DNA ArtificialRecombination region of pAd/PL DEST 148 tatttgtcta gggccgcggg gactttgaccgtttacgtgg agactcgccc aggtgttttt 60 ctcaggtgtt ttccgcgttc cgggtcaaagttggcgtttt attattatag tcagtcgaag 120 cttggatccg gtacctctag aattctcgagcggccgctag cgacatcgat cacaagtttg 180 tacaaaaaag caggctnnac ccagctttcttgtacaaagt ggtgatcgat tcgacagatc 240 actgaaatgt gtgggcgtgg cttaagggtgggaaagaata tataagg 287 149 325 DNA Artificial Recombination region ofpIB/V5 His DEST 149 cttatcgcgc ctataaatac agcccgcaac gatctggtaaacacagttga acagcatctg 60 ttcgaattta aagcttgata tcgaattcct gcagcccagcgctggatcct cgatcacaag 120 tttgtacaaa aaagcaggct nnac cca cca gct ttc ttgtac aaa gtg gtg 171 Pro Pro Ala Phe Leu Tyr Lys Val Val 1 5 atc gac ccgggt cta gag ggc ccg cgg ttc gaa ggt aag cct atc cct 219 Ile Asp Pro GlyLeu Glu Gly Pro Arg Phe Glu Gly Lys Pro Ile Pro 10 15 20 25 aac cct ctcctc ggt ctc gat tct acg cgt acc ggt cat cat cac cat 267 Asn Pro Leu LeuGly Leu Asp Ser Thr Arg Thr Gly His His His His 30 35 40 cac cat tgagtttatctga ctaaatctta gttgtattgt catgttttaa tacaatatg 325 His His 150357 DNA Artificial Recombination region of pLenti6/V5 DEST 150tcgtaacaac tccgccccat tgacgcaaat gggcggtagg cgtgtacggt gggaggtcta 60tataagcaga gctcgtttag tgaaccgtca gatcgcctgg agacgccatc cacgctgttt 120tgacctccat agaagacacc gactctagag gatccactag tccagtgtgg tggaattctg 180cagatatcaa caagtttgta caaaaaagca ggctnnac cca gct ttc ttg tac aaa 236Pro Ala Phe Leu Tyr Lys 1 5 gtg gtt gat atc cag cac agt ggc ggc cgc tcgagt cta gag ggc ccg 284 Val Val Asp Ile Gln His Ser Gly Gly Arg Ser SerLeu Glu Gly Pro 10 15 20 cgg ttc gaa ggt aag cct atc cct aac cct ctc ctcggt ctc gat tct 332 Arg Phe Glu Gly Lys Pro Ile Pro Asn Pro Leu Leu GlyLeu Asp Ser 25 30 35 acg cgt acc ggt tag taatgagttt 357 Thr Arg Thr Gly40 151 359 DNA Artificial Recombination region of the expression cloneresulting from pLenti6/UbC/V5 DEST x entry clone 151 ttggcgagtgtgttttgtga agttttttag gcaccttttg aaatgtaatc atttgggtca 60 atatgtaattttcagtgtta gactagtaaa ttgtccgcta aattctggcc gtttttggct 120 tttttgttagacgaagcttg gtaccgagct cggatccact agtccagtgt ggtggaattc 180 tgcagatatcaacaagtttg tacaaaaaag caggctnnac cca gct ttc ttg tac 235 Pro Ala Phe LeuTyr 1 5 aaa gtg gtt gat atc cag cac agt ggc ggc cgc tcg agt cta gag ggc283 Lys Val Val Asp Ile Gln His Ser Gly Gly Arg Ser Ser Leu Glu Gly 1015 20 ccg cgg ttc gaa ggt aag cct atc cct aac cct ctc ctc ggt ctc gat331 Pro Arg Phe Glu Gly Lys Pro Ile Pro Asn Pro Leu Leu Gly Leu Asp 2530 35 tct acg cgt acc ggt tag taatgagttt 359 Ser Thr Arg Thr Gly 40 152293 DNA Artificial Cloning region of pLenti6/V5 D Topo 152 tcgtaacaactccgccccat tgacgcaaat gggcggtagg cgtgtacggt gggaggtcta 60 tataagcagagctcgtttag tgaaccgtca gatcgcctgg agacgccatc cacgctgttt 120 tgacctccatagaagacacc gactctagag gatccactag tccagtgtgg tggaattgat 180 cccttcacca tgaag ggc tcg agt cta gag ggc ccg cgg ttc gaa ggt aag 231 Lys Gly Ser SerLeu Glu Gly Pro Arg Phe Glu Gly Lys 1 5 10 cct atc cct aac cct ctc ctcggt ctc gat tct acg cgt acc ggt tag 279 Pro Ile Pro Asn Pro Leu Leu GlyLeu Asp Ser Thr Arg Thr Gly 15 20 25 taatgagttt ggaa 293 153 310 DNAArtificial Recombination region of pcDNA6.2/V5 DEST 153 caaatgggcggtaggcgtgt acggtgggag gtctatataa gcagagctct ctggctaact 60 agagaacccactgcttactg gcttatcgaa attaatacga ctcactatag ggagacccaa 120 gctggctagttaagctatca acaagtttgt acaaaaaagc aggctntagn ac cca gct 178 Pro Ala 1 ttcttg tac aaa gtg gtt gat cta gag ggc ccg cgg ttc gaa ggt aag 226 Phe LeuTyr Lys Val Val Asp Leu Glu Gly Pro Arg Phe Glu Gly Lys 5 10 15 cct atccct aac cct ctc ctc ggt ctc gat tct acg cgt acc ggt tag 274 Pro Ile ProAsn Pro Leu Leu Gly Leu Asp Ser Thr Arg Thr Gly 20 25 30 taatgagtttaaacggggga ggctaactga aacacg 310 154 379 DNA Artificial Recombinationregion of pcDNA6.2/GFP DEST 154 caaatgggcg gtaggcgtgt acggtgggaggtctatataa gcagagctct ctggctaact 60 agagaaccca ctgcttactg gcttatcgaaattaatacga ctcactatag ggagacccaa 120 gctggctagt taagctatca acaagtttgtacaaaaaagc aggctntagn ac cca gct 178 Pro Ala 1 ttc ttg tac aaa gtg gttgat cta gag ggc ccc gcg gct agc aaa gga 226 Phe Leu Tyr Lys Val Val AspLeu Glu Gly Pro Ala Ala Ser Lys Gly 5 10 15 gaa gaa ctt ttc act gga ggtgtc cca att ctt gtt gaa tta gat ggt 274 Glu Glu Leu Phe Thr Gly Gly ValPro Ile Leu Val Glu Leu Asp Gly 20 25 30 gat gtt aat ggg cac aaa ttt tctgtc agt gga gag ggt gaa ggt gat 322 Asp Val Asn Gly His Lys Phe Ser ValSer Gly Glu Gly Glu Gly Asp 35 40 45 50 gct aca tac gga aag ctt acc cttaaa ttt att tgc act act gga aaa 370 Ala Thr Tyr Gly Lys Leu Thr Leu LysPhe Ile Cys Thr Thr Gly Lys 55 60 65 cta cct gtt 379 Leu Pro Val 155 5DNA Artificial Topoisomerase recognition site 155 ncctt 5 156 7 DNAArtificial Topoisomerase recognition site for type IA E. colitopoisomerase III 156 gcaactt 7 157 7 DNA Artificial Overlap region;bases 6-12 in the core region 157 tttatac 7 158 7 DNA ArtificialConsensus sequence 158 nnnatac 7 159 7 DNA Artificial Kozak consensussequence 159 nnnatgg 7 160 17 DNA Artificial Proposed Reverse PCR primersequence 160 tgagctgctg ccacaaa 17 161 7 DNA Artificial Seven base pairinverted repeat region 161 caacttt 7 162 7 DNA Artificial Seven basepair inverted repeat region 162 aaagttg 7 163 4 DNA Artificial PCRforward primer addition 163 cacc 4 164 4 DNA Artificial Overhang incloning vector 164 gtgg 4 165 7 PRT Artificial C-terminal polyhistidinetag and free carboxyl group 165 His His His His His His Xaa 1 5

What is claimed is:
 1. A nucleic acid molecule comprising all or aportion of at least one viral genome and further comprising at least tworecombination sites that do not substantially recombine with each other,wherein at least one recombination site is capable of undergoingrecombination with a compatible recombination site in the presence of atleast one protein active in lambda recombination and wherein the nucleicacid molecule replicates in prokaryotic and eukaryotic cells.
 2. Anucleic acid molecule according to claim 1, wherein the nucleic acidmolecule comprises all or a portion of at least one viral genomeselected from the group consisting of an adenovirus genome and anadeno-associate virus genome.
 3. A nucleic acid molecule according toclaim 1, wherein the nucleic acid molecule comprises all or a portion ofat least one retroviral genome.
 4. A nucleic acid molecule according toclaim 3, wherein the retroviral genome is a lentiviral genome.
 5. Anucleic acid molecule according to claim 1, wherein the nucleic acidmolecule comprises all or a portion of at least one viral genomeselected from the group consisting of a herpesvirus genome and a poxvirus genome.
 6. A nucleic acid molecule according to claim 1, whereinthe nucleic acid molecule comprises all or a portion of at least one RNAvirus genome.
 7. A nucleic acid molecule according to claim 6, whereinthe RNA virus is selected from a group consisting of a flavivirusgenome, a togavirus genome, and an alphavirus genome.
 8. A nucleic acidmolecule according to claim 1, wherein the nucleic acid molecule is aplasmid or a bacmid comprising a prokaryotic origin of replication and aselectable marker.
 9. A nucleic acid molecule according to claim 1,further comprising one or more features selected from the groupconsisting of a promoter, a viral terminal repeat, a splice site, apackaging signal, a nucleic acid sequence responsive to one or moreviral proteins, a recognition site, a recombination site, a sequencesencoding one or more marker proteins or polypeptides, a sequenceencoding one or more epitopes recognizable by an antibody, an origin ofreplication, an intervening sequence, an internal ribosome entrysequences, and a polyadenylation signal.
 10. A nucleic acid moleculeaccording to claim 1, further comprising a nucleotide sequence ofinterest between the two recombination sites.
 11. A nucleic acidmolecule according to claim 10, wherein the sequence of interestcomprises one or more sequences selected from the group consisting asequence encoding one or more polypeptides, a sequence encoding one ormore tRNA sequences, a sequence encoding one or more ribozyme sequences,one or more promoter sequences, one or more enhancer sequences, and oneor more repressor sequences.
 12. A nucleic acid molecule comprising allor a portion of a baculoviral genome and further comprising one or morerecombination sites, wherein at least one recombination site is capableof undergoing recombination with a compatible recombination site in thepresence of at least one protein active in lambda recombination.
 13. Anucleic acid molecule according to claim 12, wherein the moleculecomprises two recombination sites that do not substantially recombinewith each other.
 14. A nucleic acid molecule according to claim 13,wherein the sequence of interest comprises one or more sequencesselected from the group consisting a sequence encoding one or morepolypeptides, a sequence encoding one or more tRNA sequences, a sequenceencoding one or more ribozyme sequences, one or more promoter sequences,one or more enhancer sequences, and one or more repressor sequences. 15.A composition comprising the nucleic acid molecule of claim
 1. 16. Acomposition comprising the nucleic acid molecule of claim
 12. 17. Amethod of constructing a recombinant virus, comprising: (a) providing afirst nucleic acid molecule comprising all or a portion of at least oneviral genome and at least a first and a second recombination site thatdo not substantially recombine with each other; (b) contacting the firstnucleic acid molecule with a second nucleic acid molecule comprising asequence of interest flanked by at least a third and a fourthrecombination site under conditions such that recombination occursbetween the first and third recombination site and between the secondand fourth recombination site; and (c) introducing the nucleic acidmolecule of step (b) into a cell that packages the nucleic acid moleculeof step (b).
 18. A method according to claim 17, wherein the firstnucleic acid molecule comprises all or a portion of at least one viralgenome selected from the group consisting of an adenovirus genome and anadeno-associate virus genome.
 19. A method according to claim 17,wherein the first nucleic acid molecule comprises all or a portion of atleast one retroviral genome.
 20. A method according to claim 19, whereinthe retroviral genome is a lentiviral genome.
 21. A method according toclaim 17, wherein the first nucleic acid molecule comprises all or aportion of at least one viral genome selected from the group consistingof a herpesvirus genome and a pox virus genome.
 22. A method accordingto claim 17, wherein the first nucleic acid molecule comprises all or aportion of at least one RNA virus genome.
 23. A method according toclaim 22, wherein the RNA virus is selected from a group consisting of aflavivirus genome, a togavirus genome, and an alphavirus genome.
 24. Amethod according to claim 17, wherein the first nucleic acid molecule isa plasmid or a bacmid comprising an origin of replication and aselectable marker.
 25. A method according to claim 17, wherein theportion of the second nucleic acid between the recombination sitecomprises a nucleotide sequence of interest.
 26. A method according toclaim 25, wherein the sequence of interest comprises one or moresequences selected from a group consisting a sequence encoding one ormore polypeptides, a sequence encoding one or more tRNA sequences, asequence encoding one or more ribozyme sequences, one or more promotersequences, one or more enhancer sequences, and one or more repressorsequences.
 27. A method according to claim 17, further comprisingdigesting the first nucleic acid molecule with a restriction enzyme thatcleaves the first nucleic acid at a site between the recombinationsites.
 28. A recombinant virus produced by the method of claim
 17. 29. Acomposition comprising the recombinant virus of claim
 28. 30. A methodof producing a fusion polypeptide, comprising: providing a host cellcomprising a first nucleic acid sequence encoding the fusionpolypeptide, wherein the sequence comprises at least a first codingregion and a second coding region separated by a sequence comprising astop codon; expressing in the cell a second nucleic acid sequencecomprising one or more suppressor tRNAs that suppress the stop codon;and incubating the host cell under conditions sufficient to express thefusion polypeptide comprising the first coding region and the secondcoding region, wherein at least one of the first and/or second nucleicacid sequences is present on a nucleic acid molecule comprising all or aportion of at least one viral genome.
 31. A method according to claim30, further comprising introducing nucleic acid molecules comprising thefirst and the second nucleic acid sequences into the host cell.
 32. Amethod according to claim 30, wherein the nucleic acid moleculecomprising the second nucleic sequence comprises all or a portion of anadenoviral genome.
 33. A method according to claim 30, wherein the firstcoding region and stop codon are flanked by recombination sites that donot substantially recombine with each other.
 34. A method of expressinga polypeptide, comprising: contacting a cell with a nucleic acidmolecule comprising a sequence encoding the polypeptide operably linkedto a promoter and a repressor sequence, wherein the nucleic acidmolecule comprises all or a portion of a viral genome; contacting thecell with a nucleic acid molecule encoding a protein that binds to therepressor sequence; and incubating the cell under conditions sufficientto express the polypeptide.
 35. A method according to claim 34, whereinthe viral genome is a lentivirus genome.
 36. The method according toclaim 34, wherein the viral genome is an HIV-1 genome.
 37. The methodaccording to claim 34, wherein the repressor sequence is thetetracycline operator sequence and the protein is the tetracyclinerepressor protein.
 38. The method according to claim 37, whereinconditions sufficient to express the polypeptide comprise incubating thecell in the presence of tetracycline.
 39. A method of expressing apolypeptide, comprising: contacting a cell with a nucleic acid moleculecomprising a sequence encoding the polypeptide operably linked to apromoter and a repressor sequence, wherein the nucleic acid moleculecomprises all or a portion of a viral genome and wherein the cellexpresses a protein that binds to the repressor sequence; and incubatingthe cell under conditions sufficient to express the polypeptide.
 40. Themethod according to claim 39, wherein the viral genome is a lentiviralgenome.
 41. The method according to claim 39, wherein the viral genomeis an HIV-1 genome.
 42. The method according to claim 39, wherein therepressor sequence is the tetracycline operator sequence and the proteinis the tetracycline repressor protein.
 43. The method according to claim42, wherein conditions sufficient to express the polypeptide compriseincubating the cell in the presence of tetracycline.